Methods for the identification of inhibitors of alpha-aminoadipate reductase as antibiotics

ABSTRACT

The present inventors have discovered that α-Aminoadipate Reductase is essential for fungal pathogenicity. Specifically, the inhibition of α-Aminoadipate Reductase gene expression in fungi results in no signs of successful infection or lesions. Thus, α-Aminoadipate Reductase can be used as a target for the identification of antibiotics, preferably antifungals. Accordingly, the present invention provides methods for the identification of compounds that inhibit α-Aminoadipate Reductase expression or activity. The methods of the invention are useful for the identification of antibiotics, preferably antifungals.

FIELD OF THE INVENTION

[0001] The invention relates generally to methods for the identificationof antibiotics, preferably antifungals that affect the biosynthesis oflysine. This application is co-pending with our application entitled“Methods for the Identification of Inhibitors of Homocitrate Synthase asAntibiotics”.

BACKGROUND OF THE INVENTION

[0002] Filamentous fungi are the causal agents responsible for manyserious pathogenic infections of plants and animals. Since fungi areeukaryotes, and thus more similar to their host organisms than, forexample bacteria, the treatment of infections by fungi poses specialrisks and challenges not encountered with other types of infections. Onesuch fungus is Magnaporthe grisea, the fungus that causes rice blastdisease. It is an organism that poses a significant threat to foodsupplies worldwide. Other examples of plant pathogens of economicimportance include the pathogens in the genera Agaricus, Alternaria,Anisogramma, Anthracoidea, Antrodia, Apiognomonia, Apiosporina,Armillaria, Ascochyta, Aspergillus, Bipolaris, Bjerkandera,Botryosphaeria, Botrytis, Ceratobasidium, Ceratocystis, Cercospora,Cercosporidium, Cerotelium, Cerrena, Chondrostereum, Chryphonectria,Chrysomyxa, Cladosporium, Claviceps, Cochliobolus, Coleosporium,Colletotrichium, Colletotrichum, Corticium, Corynespora, Cronartium,Cryphonectria, Cryptosphaeria, Cyathus, Cymadothea, Cytospora,Daedaleopsis, Diaporthe, Didymella, Diplocarpon, Diplodia,Discohainesia, Discula, Dothistroma, Drechslera, Echinodontium, Elsinoe,Endocronartium, Endothia, Entyloma, Epichloe, Erysiphe, Exobasidium,Exserohilum, Fomes, Fomitopsis, Fusarium, Gaeumannomyces, Ganoderma,Gibberella, Gloeocercospora, Gloeophyllum, Gloeoporus, Glomerella,Gnomoniella, Guignardia, Gymnosporangium, Helminthosporium,Herpotrichia, Heterobasidion, Hirschioporus, Hypodermella, Inonotus,Irpex, Kabatiella, Kabatina, Laetiporus, Laetisaria, Lasiodiplodia,Laxitextum, Leptographium, Leptosphaeria, Leptosphaerulina,Leucytospora, Linospora, Lophodermella, Lophodermium, Macrophomina,Magnaporthe, Marssonina, Melampsora, Melampsorella, Meria, Microdochium,Microsphaera, Monilinia, Monochaetia, Morchella, Mycosphaerella,Myrothecium, Nectria, Nigrospora, Ophiosphaerella, Ophiostoma,Penicillium, Perenniporia, Peridermium, Pestalotia, Phaeocryptopus,Phaeolus, Phakopsora, Phellinus, Phialophora, Phoma, Phomopsis,Phragmidium, Phyllachora, Phyllactinia, Phyllosticta, Phymatotrichopsis,Pleospora, Podosphaera, Pseudopeziza, Pseudoseptoria, Puccinia,Pucciniastrum, Pyricularia, Rhabdocline, Rhizoctonia, Rhizopus,Rhizosphaera, Rhynchosporium, Rhytisma, Schizophyllum, Schizopora,Scirrhia, Sclerotinia, Sclerotium, Scytinostroma, Septoria, Setosphaera,Sirococcus, Spaerotheca, Sphaeropsis, Sphaerotheca, Sporisorium,Stagonospora, Stemphylium, Stenocarpella, Stereum, Taphrina,Thielaviopsis, Tilletia, Trametes, Tranzschelia, Trichoderma, Tubakia,Typhula, Uncinula, Urocystis, Uromyces, Ustilago, Valsa, Venturia,Verticillium, Xylaria, and others. Related organisms in theclassification, oomycetes, that include the genera Albugo, Aphanomyces,Bremia, Peronospora, Phytophthora, Plasmodiophora, Plasmopara,Pseudoperonospora, Pythium, Sclerophthora, and others are alsosignificant plant pathogens and are sometimes classified along with thetrue fungi. Human diseases that are caused by filamentous fungi includelife-threatening lung and disseminated diseases, often a result ofinfections by Aspergillus fumigatus. Other fungal diseases in animalsare caused by fungi in the genera, Fusarium, Blastomyces, Microsporum,Trichophyton, Epidermophyton, Candida, Histoplamsa, Pneumocystis,Cryptococcus, other Aspergilli, and others. The control of fungaldiseases in plants and animals is usually mediated by chemicals thatinhibit the growth, proliferation, and/or pathogenicity of the fungalorganisms. To date, there are less than twenty known modes-of-action forplant protection fungicides and human antifungal compounds.

[0003] A pathogenic organism has been defined as an organism thatcauses, or is capable of causing disease. Pathogenic organisms propagateon or in tissues and may obtain nutrients and other essential materialsfrom their hosts. A substantial amount of work concerning filamentousfungal pathogens has been performed with the human pathogen, Aspergillusfumigatus. Shibuya et al. (Shibuya, K., M. Takaoka, et al (1999) MicrobPathog 27: 123-31 (PMID: 10455003)) have shown that the deletion ofeither of two suspected pathogenicity related genes encoding an alkalineprotease or a hydrophobin (rodlet) respectively, did not reducemortality of mice infected with these mutant strains. Smith et al.(Smith, J. M., C. M. Tang, et al. (1994) Infect Immun 62: 5247-54 (PMID:7960101)) showed similar results with alkaline protease and theribotoxin restrictocin; Aspergillus fumigatus strains mutated for eitherof these genes were fully pathogenic to mice. Reichard et al. (Reichard,U., M. Monod, et al. (1997) J Med Vet Mycol 35: 189-96 (PMID: 9229335))showed that deletion of the suspected pathogenicity gene encoding,aspergillopepsin (PEP) in Aspergillus fumigatus, had no effect onmortality in a guinea pig model system, and Aufauvre-Brown et al(Aufauvre-Brown, A., E. Mellado, et al. (1997) Fungal Genet Biol 21:141-52 (PMID: 9073488)) showed no effects of a chitin synthase mutationon pathogenicity. However, not all experiments produced negativeresults. Ergosterol is an important membrane component found in fungalorganisms. Pathogenic fungi that lack key enzymes in this biochemicalpathway might be expected to be non-pathogenic since neither the plantnor animal hosts contain this particular sterol. Many antifungalcompounds that affect this biochemical pathway have been described(Onishi, J. C. and A. A. Patchett (1990a, b, c, d, and e) U.S. Pat. Nos.4,920,109; 4,920,111; 4,920,112; 4,920,113; and 4,921,844, Merck & Co.Inc. (Rahway N.J.)) and (Hewitt, H. G. (1998) Fungicides in CropProtection Cambridge, University Press). D'Enfert et al. (D'Enfert, C.,M. Diaquin, et al. (1996) Infect Immun 64: 4401-5 (PMID: 8926121))showed that an Aspergillus fumigatus strain mutated in an orotidine5′-phosphate decarboxylase gene was entirely non-pathogenic in mice, andBrown et al. (Brown, J. S., A. Aufauvre-Brown, et al. (2000) MolMicrobiol 36:1371-80 (PMID: 10931287)) observed a non-pathogenic resultwhen genes involved in the synthesis of para-aminobenzoic acid weremutated. Some specific target genes have been described as havingutility for the screening of inhibitors of plant pathogenic fungi. Bacotet al. (Bacot, K. O., D. B. Jordan, et al. (2000) U.S. Pat. No.6,074,830, E. I. du Pont de Nemours & Company (Wilmington Del.))describe the use of 3,4-dihydroxy-2-butanone 4-phosphate synthase, andDavis et al. (Davis, G. E., G. D. Gustafson, et al. (1999) U.S. Pat. No.5,976,848, Dow AgroSciences LLC (Indianapolis Ind.)) describe the use ofdihydroorotate dehydrogenase for potential screening purposes.

[0004] There are also a number of papers that report less clear results,showing neither fill pathogenicity nor non-pathogenicity of mutants.Hensel et al. (Hensel, M., H. N. Arst, Jr., et al. (1998) Mol Gen Genet258: 553-7 (PMID: 9669338)) showed only moderate effects of the deletionof the areA transcriptional activator on the pathogenicity ofAspergillus fumigatus. Tang et al. (Tang, C. M., J. M. Smith, et al.(1994) Infect Immun 62: 5255-60 (PMID: 7960102)) using the relatedfungus, Aspergillus nidulans, observed that a mutation inpara-aminobenzoic acid synthesis prevented mortality in mice, while amutation in lysine biosynthesis had no significant effect on themortality of the infected mice.

[0005] Therefore, it is not currently possible to determine whichspecific growth materials may be readily obtained by a pathogen from itshost, and which materials may not. Surprising, especially in light ofthe results showing that a lysine biosynthesis mutation in thefilamentous fungus, Aspergillus nidulans, had no significant effect onthe pathogenicity in a mouse model system (Tang, C. M., J. M. Smith, etal. (1994) Infect Immun 62: 5255-60 (PMID: 7960102)), we have found thatMagnaporthe grisea that cannot synthesize their own lysine are entirelynon-pathogenic on their host organism. To date there do not appear to beany publications demonstrating an anti-pathogenic effect of theknock-out, over-expression, antisense expression, or inhibition of thegenes or gene products involved in lysine biosynthesis in filamentousfungi. Thus, it has not been shown that the de novo biosynthesis oflysine is essential for fungal pathogenicity. Our co-pending applicationentitled “Methods for the Identification of Inhibitors of HomocitrateSynthase as Antibiotics” shows that the disruption of lysinebiosynthesis as the result of a disruption of the gene encoding theenzyme activity, homocitrate synthase, also results in a non-pathogenicphenotype for M. grisea. Thus, it would be desirable to determine theutility of the enzymes involved in lysine biosynthesis for evaluatingantibiotic compounds, especially fungicides. If a fungal biochemicalpathway or specific gene product in that pathway is shown to be requiredfor fungal pathogenicity, various formats of in vitro and in vivoscreening assays may be put in place to discover classes of chemicalcompounds that react with the validated target gene, gene product, orbiochemical pathway, and are thus candidates for antifungal, biocide,and biostatic materials.

SUMMARY OF THE INVENTION

[0006] Surprisingly, the present inventors have discovered that in vivodisruption of the gene encoding α-Aminoadipate Reductase in Magnaporthegrisea prevents or inhibits the pathogenicity of the fungus. Thus, thepresent inventors have discovered that α-Aminoadipate Reductase isessential for normal rice blast pathogenicity, and can be used as atarget for the identification of antibiotics, preferably fungicides.Accordingly, the present invention provides methods for theidentification of compounds that inhibit α-Aminoadipate Reductaseexpression or activity. The methods of the invention are useful for theidentification of antibiotics, preferably fungicides.

BRIEF DESCRIPTION OF THE FIGURES

[0007]FIG. 1 shows the reaction performed by α-Aminoadipate Reductase(AAR1) reaction. The Substrates/Products are L-2-Aminoadipate+NADPH+ATPand the Products/Substrates are L-2-Aminoadipate6-semialdehyde+NADP++AMP+pyrophosphate+H₂O. The function of theα-Aminoadipate Reductase enzyme is the interconversion ofL-2-Aminoadipate, NADPH, and ATP to L-2-Aminoadipate 6-semialdehyde,NADP+, AMP, pyrophosphate, and H₂O. This reaction is part of the lysinebiosynthesis pathway.

[0008]FIG. 2 shows a digital image showing the effect of AAR1 genedisruption on Magnaporthe grisea pathogenicity using whole plantinfection assays. Rice variety C039 was inoculated with wild-type andthe transposon insertion strains, KO1-1 and KO1-11. Leaf segments wereimaged at five days post-inoculation.

[0009]FIG. 3. Verification of Gene Function by Analysis of NutritionalRequirements. Wild-type and transposon insertion strains, KO1-1 andKO1-11, were grown in (A) minimal media and (B) minimal media with theaddition of L-lysine, respectively. The x-axis shows time in days andthe y-axis shows turbidity measured at 490 nanometers and 750nanometers. The symbols represent wildtype (--♦--), transposon strainKO1-1 (--▪--), and transposon strain KO1-11 (--▴--).

DETAILED DESCRIPTION OF THE INVENTION

[0010] Unless otherwise indicated, the following terms are intended tohave the following meanings in interpreting the present invention.

[0011] The term “active against” in the context of compounds, agents, orcompositions having antibiotic activity indicates that the compoundexerts an effect on a particular target or targets which is deleteriousto the in vitro and/or in vivo growth of an organism having that targetor targets. In particular, a compound active against a gene exerts anaction on a target which affects an expression product of that gene.This does not necessarily mean that the compound acts directly on theexpression product of the gene, but instead indicates that the compoundaffects the expression product in a deleterious manner. Thus, the directtarget of the compound may be, for example, at an upstream componentwhich reduces transcription from the gene, resulting in a lower level ofexpression. Likewise, the compound may affect the level of translationof a polypeptide expression product, or may act on a downstreamcomponent of a biochemical pathway in which the expression product ofthe gene has a major biological role. Consequently, such a compound canbe said to be active against the gene, against the gene product, oragainst the related component either upstream or downstream of that geneor expression product. While the term “active against” encompasses abroad range of potential activities, it also implies some degree ofspecificity of target. Therefore, for example, a general protease is not“active against” a particular gene which produces a polypeptide product.In contrast, a compound which inhibits a particular enzyme is activeagainst that enzyme and against the gene which codes for that enzyme.

[0012] As used herein, the term “allele” refers to any of thealternative forms of a gene that may occur at a given locus.

[0013] As used herein, the term “α-Aminoadipate Reductase (EC 1.2.1.31),α-Aminoadipate Reductase polypeptide, α-Aminoadipate semialdehydedehydrogenase, or Alpha-Aminoadipate Reductase” is synonymous with “theAAR1 gene product” or the “the ASD1 gene product” and refers to anenzyme that catalyses the interconversion of L-2-Aminoadipate, NADPH,and ATP with L-2-Aminoadipate 6-semialdehyde, NADP+, AMP, pyrophosphate,and H₂O.

[0014] The term “antibiotic” refers to any substance or compound thatwhen contacted with a living cell, organism, virus, or other entitycapable of replication, results in a reduction of growth, viability, orpathogenicity of that entity.

[0015] The term “binding” refers to a non-covalent or a covalentinteraction, preferably non-covalent, that holds two molecules together.For example, two such molecules could be an enzyme and an inhibitor ofthat enzyme. Non-covalent interactions include hydrogen bonding, ionicinteractions among charged groups, van der Waals interactions andhydrophobic interactions among nonpolar groups. One or more of theseinteractions can mediate the binding of two molecules to each other.

[0016] The term “biochemical pathway” or “pathway” refers to a connectedseries of biochemical reactions normally occurring in a cell, or morebroadly a cellular event such as cellular division or DNA replication.Typically, the steps in such a biochemical pathway act in a coordinatedfashion to produce a specific product or products or to produce someother particular biochemical action. Such a biochemical pathway requiresthe expression product of a gene if the absence of that expressionproduct either directly or indirectly prevents the completion of one ormore steps in that pathway, thereby preventing or significantly reducingthe production of one or more normal products or effects of thatpathway. Thus, an agent specifically inhibits such a biochemical pathwayrequiring the expression product of a particular gene if the presence ofthe agent stops or substantially reduces the completion of the series ofsteps in that pathway. Such an agent, may, but does not necessarily, actdirectly on the expression product of that particular gene.

[0017] As used herein, the term “cDNA” means complementarydeoxyribonucleic acid.

[0018] As used herein, the term “CoA” means coenzyme A.

[0019] As used herein, the term “conditional lethal” refers to amutation permitting growth and/or survival only under special growth orenvironmental conditions.

[0020] As used herein, the term “cosmid” refers to a hybrid vector, usedin gene cloning, that includes a cos site (from the lambdabacteriophage). It also contains drug resistance marker genes and otherplasmid genes. Cosmids are especially suitable for cloning large genesor multigene fragments.

[0021] As used herein, the term “dominant allele” refers to a dominantmutant allele in which a discernable mutant phenotype can be detectedwhen this mutation is present in an organism that also contains a wildtype (non-mutant), recessive allele, or other dominant allele.

[0022] As used herein, the term “DNA” means deoxyribonucleic acid.

[0023] As used herein, the term “ELISA” means enzyme-linkedimmunosorbent assay.

[0024] “Fungi” (singular: fungus) refers to whole fungi, fungal organsand tissues (e.g., asci, hyphae, pseudohyphae, rhizoid, sclerotia,sterigmata, spores, sporodochia, sporangia, synnemata, conidia,ascostroma, cleistothecia, mycelia, perithecia, basidia and the like),spores, fungal cells and the progeny thereof. Fungi are a group oforganisms (about 50,000 known species), including, but not limited to,mushrooms, mildews, moulds, yeasts, etc., comprising the kingdom Fungi.They can either exist as single cells or make up a multicellular bodycalled a mycelium, which consists of filaments known as hyphae. Mostfungal cells are multinucleate and have cell walls, composed chiefly ofchitin. Fungi exist primarily in damp situations on land and, because ofthe absence of chlorophyll and thus the inability to manufacture theirown food by photosynthesis, are either parasites on other organisms orsaprotrophs feeding on dead organic matter. The principal criteria usedin classification are the nature of the spores produced and the presenceor absence of cross walls within the hyphae. Fungi are distributedworldwide in terrestrial, freshwater, and marine habitats. Some live inthe soil. Many pathogenic fungi cause disease in animals and man or inplants, while some saprotrophs are destructive to timber, textiles, andother materials. Some fungi form associations with other organisms, mostnotably with algae to form lichens.

[0025] As used herein, the term “fungicide”, “antifungal”, or“antimycotic” refers to an antibiotic substance or compound that killsor suppresses the growth, viability, or pathogenicity of at least onefungus, fungal cell, fungal tissue or spore.

[0026] In the context of this disclosure, “gene” should be understood torefer to a unit of heredity. Each gene is composed of a linear chain ofdeoxyribonucleotides which can be referred to by the sequence ofnucleotides forming the chain. Thus, “sequence” is used to indicate boththe ordered listing of the nucleotides which form the chain, and thechain, itself, which has that sequence of nucleotides. (“Sequence” isused in the similar way in referring to RNA chains, linear chains madeof ribonucleotides.) The gene may include regulatory and controlsequences, sequences which can be transcribed into an RNA molecule, andmay contain sequences with unknown function. The majority of the RNAtranscription products are messenger RNAs (mRNAs), which includesequences which are translated into polypeptides and may includesequences which are not translated. It should be recognized that smalldifferences in nucleotide sequence for the same gene can exist betweendifferent fungal strains, or even within a particular fungal strain,without altering the identity of the gene.

[0027] As used in this disclosure, the terms “growth” or “cell growth”of an organism refers to an increase in mass, density, or number ofcells of said organism. Some common methods for the measurement ofgrowth include the determination of the optical density of a cellsuspension, the counting of the number of cells in a fixed volume, thecounting of the number of cells by measurement of cell division, themeasurement of cellular mass or cellular volume, and the like.

[0028] As used in this disclosure, the term “growth conditionalphenotype” indicates that a fungal strain having such a phenotypeexhibits a significantly greater difference in growth rates in responseto a change in one or more of the culture parameters than an otherwisesimilar strain not having a growth conditional phenotype. Typically, agrowth conditional phenotype is described with respect to a singlegrowth culture parameter, such as temperature. Thus, a temperature (orheat-sensitive) mutant (i.e., a fungal strain having a heat-sensitivephenotype) exhibits significantly different growth, and preferably nogrowth, under non-permissive temperature conditions as compared togrowth under permissive conditions. In addition, such mutants preferablyalso show intermediate growth rates at intermediate, or semi-permissive,temperatures. Similar responses also result from the appropriate growthchanges for other types of growth conditional phenotypes.

[0029] As used herein, the term “H₂O” means water.

[0030] As used herein, the term “AAR1” means a gene encoding aAminoadipate Reductase activity, referring to an enzyme that catalysesthe interconversion of L-2-Aminoadipate, NADPH, and ATP withL-2-Aminoadipate 6-semialdehyde, NADP+, AMP, pyrophosphate, and H₂O.

[0031] As used herein, the term “heterologous AAR1 gene” means a gene,not derived from Magnaporthe grisea, and having: at least 50% sequenceidentity, preferably 60%, 70%, 80%, 90%, 95%, 99% sequence identity andeach integer unit of sequence identity from 50-100% in ascending orderto SEQ ID NO: 1 or SEQ ID NO: 2; or at least 10% of the activity of aMagnaporthe grisea α-Aminoadipate Reductase, preferably 25%, 50%, 75%,90%, 95%, 99% and each integer unit of activity from 10-100% inascending order.

[0032] As used herein, the term “His-Tag” refers to an encodedpolypeptide consisting of multiple consecutive histidine amino acids.

[0033] As used herein, the term “HPLC” means high pressure liquidchromatography.

[0034] As used herein, the terms “hph”, “hygromycin Bphosphotransferase”, and “hygromycin resistance gene” refer to the E.coli hygromycin phosphotransferase gene or gene product.

[0035] As used herein, the term “hygromycin B” refers to anaminoglycosidic antibiotic, used for selection and maintenance ofeukaryotic cells containing the E. coli hygromycin resistance gene.

[0036] “Hypersensitive” refers to a phenotype in which cells are moresensitive to antibiotic compounds than are wild-type cells of similar oridentical genetic background.

[0037] “Hyposensitive” refers to a phenotype in which cells are lesssensitive to antibiotic compounds than are wild-type cells of similar oridentical genetic background.

[0038] As used herein, the term “imperfect state” refers to aclassification of a fungal organism having no demonstrable sexual lifestage.

[0039] The term “inhibitor”, as used herein, refers to a chemicalsubstance that inactivates the enzymatic activity of α-AminoadipateReductase or substantially reduces the level of enzymatic activity,wherein “substantially” means a reduction at least as great as thestandard deviation for a measurement, preferably a reduction by 50%,more preferably a reduction of at least one magnitude, i.e. to 10%. Theinhibitor may function by interacting directly with the enzyme, acofactor of the enzyme, the substrate of the enzyme, or any combinationthereof.

[0040] A polynucleotide may be “introduced” into a fungal cell by anymeans known to those of skill in the art, including transfection,transformation or transduction, transposable element, electroporation,particle bombardment, infection and the like. The introducedpolynucleotide may be maintained in the cell stably if it isincorporated into a non-chromosomal autonomous replicon or integratedinto the fungal chromosome. Alternatively, the introduced polynucleotidemay be present on an extra-chromosomal non-replicating vector and betransiently expressed or transiently active.

[0041] As used herein, the term “knockout” or “gene disruption” refersto the creation of organisms carrying a null mutation (a mutation inwhich there is no active gene product), a partial null mutation ormutations, or an alteration or alterations in gene regulation byinterrupting a DNA sequence through insertion of a foreign piece of DNA.Usually the foreign DNA encodes a selectable marker.

[0042] As used herein, the term “LB agar” means Luria's Broth agar.

[0043] The term “method of screening” means that the method is suitable,and is typically used, for testing for a particular property or effectin a large number of compounds. Typically, more than one compound istested simultaneously (as in a 96-well microtiter plate), and preferablysignificant portions of the procedure can be automated. “method ofscreening” also refers to determining a set of different properties oreffects of one compound simultaneously.

[0044] As used herein, the term “mRNA” means messenger ribonucleic acid.

[0045] As used herein, the term “mutant form” of a gene refers to a genewhich has been altered, either naturally or artificially, changing thebase sequence of the gene. The change in the base sequence may be ofseveral different types, including changes of one or more bases fordifferent bases, deletions, and/or insertions, such as by a transposon.By contrast, a normal form of a gene (wild type) is a form commonlyfound in natural populations of an organism. Commonly a single form of agene will predominate in natural populations. In general, such a gene issuitable as a normal form of a gene, however, other forms which providesimilar functional characteristics may also be used as a normal gene. Inparticular, a normal form of a gene does not confer a growth conditionalphenotype on the strain having that gene, while a mutant form of a genesuitable for use in these methods does provide such a growth conditionalphenotype.

[0046] As used herein, the term “Ni” refers to nickel.

[0047] As used herein, the term “Ni-NTA” refers to nickel sepharose.

[0048] As used herein, the term “one form” of a gene is synonymous withthe term “gene”, and a “different form” of a gene refers to a gene thathas greater than 49% sequence identity and less than 100% sequenceidentity with said first form.

[0049] As used herein, the term “pathogenicity” refers to a capabilityof causing disease. The term is applied to parasitic microorganisms inrelation to their hosts.

[0050] As used herein, the term “PCR” means polymerase chain reaction.

[0051] The “percent (%) sequence identity” between two polynucleotide ortwo polypeptide sequences is determined according to the either theBLAST program (Basic Local Alignment Search Tool; (Altschul, S. F., W.Gish, et al. (1990) J Mol Biol 215: 403-10 (PMID: 2231712)) at theNational Center for Biotechnology or using Smith Waterman Alignment(Smith, T. F. and M. S. Waterman (1981) J Mol Biol 147: 195-7 (PMID:7265238)) as incorporated into GeneMatcher Plus™. It is understood thatfor the purposes of determining sequence identity when comparing a DNAsequence to an RNA sequence, a thymine nucleotide is equivalent to auracil nucleotide.

[0052] By “polypeptide” is meant a chain of at least two amino acidsjoined by peptide bonds. The chain may be linear, branched, circular orcombinations thereof. Preferably, polypeptides are from about 10 toabout 1000 amino acids in length, more preferably 10-50 amino acids inlength. The polypeptides may contain amino acid analogs and othermodifications, including, but not limited to glycosylated orphosphorylated residues.

[0053] As used herein, the term “proliferation” is synonymous to theterm “growth”.

[0054] As used herein, the term “reverse transcriptase-PCR” meansreverse transcription-polymerase chain reaction.

[0055] As used herein, the term “RNA” means ribonucleic acid.

[0056] As used herein, “semi-permissive conditions” are conditions inwhich the relevant culture parameter for a particular growth conditionalphenotype is intermediate between permissive conditions andnon-permissive conditions. Consequently, in semi-permissive conditionsan organism having a growth conditional phenotype will exhibit growthrates intermediate between those shown in permissive conditions andnon-permissive conditions. In general, such intermediate growth rate maybe due to a mutant cellular component which is partially functionalunder semi-permissive conditions, essentially fully functional underpermissive conditions, and is non-functional or has very low functionunder non-permissive conditions, where the level of function of thatcomponent is related to the growth rate of the organism. An intermediategrowth rate may also be a result of a nutrient substance or substancesthat are present in amounts not sufficient for optimal growth rates tobe achieved.

[0057] “Sensitivity phenotype” refers to a phenotype that exhibitseither hypersensitivity or hyposensitivity.

[0058] The term “specific binding” refers to an interaction betweenα-Aminoadipate Reductase and a molecule or compound, wherein theinteraction is dependent upon the primary amino acid sequence and/or theconformation of α-Aminoadipate Reductase.

[0059] As used herein, the term “TLC” means thin layer chromatography.

[0060] “Transform”, as used herein, refers to the introduction of apolynucleotide (single or double stranded DNA, RNA, or a combinationthereof) into a living cell by any means. Transformation may beaccomplished by a variety of methods, including, but not limited to,electroporation, polyethylene glycol mediated uptake, particlebombardment, agrotransformation, and the like. This process may resultin transient or stable expression of the transformed polynucleotide. By“stably transformed” is meant that the sequence of interest isintegrated into a replicon in the cell, such as a chromosome or episome.Transformed cells encompass not only the end product of a transformationprocess, but also the progeny thereof which retain the polynucleotide ofinterest.

[0061] For the purposes of the invention, “transgenic” refers to anycell, spore, tissue or part, that contains all or part of at least onerecombinant polynucleotide. In many cases, all or part of therecombinant polynucleotide is stably integrated into a chromosome orstable extra-chromosomal element, so that it is passed on to successivegenerations.

[0062] As used herein, the term “transposase” refers to an enzyme thatcatalyzes transposition. Preferred transposons are described in WO00/55346, PCT/US00/07317, and U.S. Ser. No. 09/658859.

[0063] As used herein, the term “transposition” refers to a complexgenetic rearrangement process involving the movement or copying of apolynucleotide (transposon) from one location and insertion intoanother, often within or between a genome or genomes, or DNA constructssuch as plasmids, bacmids, and cosmids.

[0064] As used herein, the term “transposon” (also known as a“transposable element”, “transposable genetic element”, “mobileelement”, or “jumping gene”) refers to a mobile DNA element such asthose, for example, described in WO00/55346, PCT/US00/07317, and U.S.Ser. No. 09/658859. Transposons can disrupt gene expression or causedeletions and inversions, and hence affect both the genotype andphenotype of the organisms concerned. The mobility of transposableelements has long been used in genetic manipulation, to introduce genesor other information into the genome of certain model systems.

[0065] As used herein, the term “Tween 20” means sorbitanmono-9-octadecenoate poly(oxy-1,1-ethanediyl).

[0066] As used in this disclosure, the term “viability” of an organismrefers to the ability of an organism to demonstrate growth underconditions appropriate for said organism, or to demonstrate an activecellular function. Some examples of active cellular functions includerespiration as measured by gas evolution, secretion of proteins and/orother compounds, dye exclusion, mobility, dye oxidation, dye reduction,pigment production, changes in medium acidity, and the like.

[0067] The present inventors have discovered that disruption of the AAR1gene and/or gene product inhibits the pathogenicity of Magnaporthegrisea. Thus, the inventors are the first to demonstrate thatα-Aminoadipate Reductase is a target for antibiotics, preferablyantifungals.

[0068] Accordingly, the invention provides methods for identifyingcompounds that inhibit AAR1 gene expression or biological activity ofits gene product(s). Such methods include ligand binding assays, assaysfor enzyme activity, cell-based assays, and assays for AAR1 geneexpression. Any compound that is a ligand for α-Aminoadipate Reductasemay have antibiotic activity. For the purposes of the invention,“ligand” refers to a molecule that will bind to a site on a polypeptide.The compounds identified by the methods of the invention are useful asantibiotics.

[0069] Thus, in one embodiment, the invention provides a method foridentifying a test compound as a candidate for an antibiotic,comprising:

[0070] a) contacting a α-Aminoadipate Reductase polypeptide with saidtest compound; and

[0071] b) detecting the presence or absence of binding between said testcompound and said α-Aminoadipate Reductase polypeptide;

[0072] wherein binding indicates that said test compound is a candidatefor an antibiotic.

[0073] The α-Aminoadipate Reductase protein may have the amino acidsequence of a naturally occurring α-Aminoadipate Reductase found in afungus, animal, plant, or microorganism, or may have an amino acidsequence derived from a naturally occurring sequence. Preferably theα-Aminoadipate Reductase is a fungal α-Aminoadipate Reductase. The cDNA(SEQ ID NO: 1) encoding the α-Aminoadipate Reductase protein, thegenomic DNA (SEQ ID NO: 2) encoding the protein, and the polypeptide(SEQ ID NO: 3) can be found herein.

[0074] By “fungal α-Aminoadipate Reductase” is meant an enzyme that canbe found in at least one fungus, and which catalyzes the interconversionof L-2-Aminoadipate and NADPH and ATP with L-2-Aminoadipate6-semialdehyde, NADP+, AMP, pyrophosphate, and H₂O. The α-AminoadipateReductase may be from any of the fungi, including ascomycota,zygomycota, basidiomycota, chytridiomycota, and lichens.

[0075] In one embodiment, the α-Aminoadipate Reductase is a Magnaportheα-Aminoadipate Reductase. Magnaporthe species include, but are notlimited to, Magnaporthe rhizophila, Magnaporthe salvinii, Magnaporthegrisea and Magnaporthe poae and the imperfect states of Magnaporthe inthe genus Pyricularia. Preferably, the Magnaporthe α-AminoadipateReductase is from Magnaporthe grisea.

[0076] In various embodiments, the α-Aminoadipate Reductase can be fromPowdery Scab (Spongospora subterranea), Grey Mould (Botrytis cinerea),White Rot (Armillaria mellea), Heartrot Fungus (Ganoderma adspersum),Brown-Rot (Piptoporus betulinus), Corn Smut (Ustilago maydis), Heartrot(Polyporus squamosus), Gray Leaf Spot (Cercospora zeae-maydis), HoneyFungus (Armillaria gallica), Root rot (Armillaria luteobubalina),Shoestring Rot (Armillaria ostoyae), Banana Anthracnose Fungus(Colletotrichum musae), Apple-rotting Fungus (Monilinia fructigena),Apple-rotting Fungus (Penicillium expansum), Clubroot Disease(Plasmodiophora brassicae), Potato Blight (Phytophthora infestans), Rootpathogen (Heterobasidion annosum), Take-all Fungus (Gaeumannomycesgraminis), Dutch Elm Disease (Ophiostoma ulmi), Bean Rust (Uromycesappendiculatus), Northern Leaf Spot (Cochliobolus carbonum), MiloDisease (Periconia circinata), Southern Corn Blight (Cochliobolusheterostrophus), Leaf Spot (Cochliobolus lunata), Brown Stripe(Cochliobolus stenospilus), Panama disease (Fusarium oxysporum), WheatHead Scab Fungus (Fusarium graminearum), Cereal Foot Rot (Fusariumculmorum), Potato Black Scurf (Rhizoctonia solani), Wheat Black StemRust (Puccinia graminis), White mold (Sclerotinia sclerotiorum), and thelike.

[0077] Fragments of an α-Aminoadipate Reductase polypeptide may be usedin the methods of the invention, preferably if the fragments include anintact or nearly intact epitope that occurs on the biologically activewildtype α-Aminoadipate Reductase. The fragments comprise at least 10consecutive amino acids of an α-Aminoadipate Reductase. Preferably, thefragment comprises at least 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90,100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230,240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370,380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510,520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650,660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790,800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930,940, 950, 960, 970, 980, 990, 1000, 1010, 1020, 1030, 1040, 1050, 1060,1070, 1080, 1090, 1100, 1110, 1120, 1130, 1140, 1150, 1160, 1170, or atleast 1180 consecutive amino acids residues of an α-AminoadipateReductase. In one embodiment, the fragment is from a Magnaportheα-Aminoadipate Reductase. Preferably, the fragment contains an aminoacid sequence conserved among fungal α-Aminoadipate Reductases.

[0078] Polypeptides having at least 50% sequence identity with a fungalα-Aminoadipate Reductase are also useful in the methods of theinvention. Preferably, the sequence identity is at least 60%, morepreferably the sequence identity is at least 70%, most preferably thesequence identity is at least 80% or 90 or 95 or 99%, or any integerfrom 60-100% sequence identity in ascending order.

[0079] In addition, it is preferred that the polypeptide has at least10% of the activity of a fungal α-Aminoadipate Reductase. Morepreferably, the polypeptide has at least 25%, at least 50%, at least 75%or at least 90% of the activity of a fungal α-Aminoadipate Reductase.Most preferably, the polypeptide has at least 10%, at least 25%, atleast 50%, at least 75% or at least 90% of the activity of the M. griseaα-Aminoadipate Reductase protein.

[0080] Thus, in another embodiment, the invention provides a method foridentifying a test compound as a candidate for a fungicide, comprising:

[0081] a) contacting said test compound with at least one polypeptideselected from the group consisting of: a polypeptide having at least tenconsecutive amino acids of a fungal α-Aminoadipate Reductase, apolypeptide having at least 50% sequence identity with a fungalα-Aminoadipate Reductase, and a polypeptide having at least 10% of theactivity thereof; and

[0082] b) detecting the presence and/or absence of binding between saidtest compound and said polypeptide;

[0083] wherein binding indicates that said test compound is a candidatefor an antibiotic.

[0084] Any technique for detecting the binding of a ligand to its targetmay be used in the methods of the invention. For example, the ligand andtarget are combined in a buffer. Many methods for detecting the bindingof a ligand to its target are known in the art, and include, but are notlimited to the detection of an immobilized ligand-target complex or thedetection of a change in the properties of a target when it is bound toa ligand. For example, in one embodiment, an array of immobilizedcandidate ligands is provided. The immobilized ligands are contactedwith an α-Aminoadipate Reductase protein or a fragment or variantthereof, the unbound protein is removed and the bound α-AminoadipateReductase is detected. In a preferred embodiment, bound α-AminoadipateReductase is detected using a labeled binding partner, such as a labeledantibody. In a variation of this assay, α-Aminoadipate Reductase islabeled prior to contacting the immobilized candidate ligands. Preferredlabels include fluorescent or radioactive moieties. Preferred detectionmethods include fluorescence correlation spectroscopy (FCS) andFCS-related confocal nanofluorimetric methods.

[0085] Once a compound is identified as a candidate for an antibiotic,it can be tested for the ability to inhibit α-Aminoadipate Reductaseenzymatic activity. The compounds can be tested using either in vitro orcell based assays. Alternatively, a compound can be tested by applyingit directly to a fungus or fungal cell, or expressing it therein, andmonitoring the fungus or fungal cell for changes or decreases in growth,development, viability, pathogenicity, or alterations in geneexpression. Thus, in one embodiment, the invention provides a method fordetermining whether a compound identified as an antibiotic candidate byan above method has antifungal activity, further comprising: contactinga fungus or fungal cells with said antifungal candidate and detecting adecrease in the growth, viability, or pathogenicity of said fungus orfungal cells.

[0086] By decrease in growth, is meant that the antifungal candidatecauses at least a 10% decrease in the growth of the fungus or fungalcells, as compared to the growth of the fungus or fungal cells in theabsence of the antifungal candidate. By a decrease in viability is meantthat at least 20% of the fungal cells, or portion of the funguscontacted with the antifungal candidate are nonviable. Preferably, thegrowth or viability will be decreased by at least 40%. More preferably,the growth or viability will be decreased by at least 50%, 75% or atleast 90% or more. Methods for measuring fungal growth and cellviability are known to those skilled in the art. By decrease inpathogenicity, is meant that the antifungal candidate causes at least a10% decrease in the disease caused by contact of the fungal pathogenwith its host, as compared to the disease caused in the absence of theantifungal candidate. Preferably, the disease will be decreased by atleast 40%. More preferably, the disease will be decreased by at least50%, 75% or at least 90% or more. Methods for measuring fungal diseaseare well known to those skilled in the art, and include such metrics aslesion formation, lesion size, sporulation, respiratory failure, and/ordeath.

[0087] The ability of a compound to inhibit α-Aminoadipate Reductaseactivity can be detected using in vitro enzymatic assays in which thedisappearance of a substrate or the appearance of a product is directlyor indirectly detected. α-Aminoadipate Reductase catalyzes theirreversible or reversible reaction L-2-Aminoadipate and NADPH andATP=L-2-Aminoadipate 6-semialdehyde, NADP+, AMP, pyrophosphate, and H₂O(see FIG. 1). Methods for detection of L-2-Aminoadipate,L-2-Aminoadipate 6-semialdehyde, NADP+, NADPH, AMP, ATP, H₂O and/orpyrophosphate, include spectrophotometry, mass spectroscopy, thin layerchromatography (TLC) and reverse phase HPLC.

[0088] Thus, the invention provides a method for identifying a testcompound as a candidate for an antibiotic, comprising either:

[0089] a) contacting L-2-Aminoadipate and NADPH and ATP with anα-Aminoadipate Reductase;

[0090] b) contacting L-2-Aminoadipate and NADPH and ATP withα-Aminoadipate Reductase and said test compound; and

[0091] c) determining the change in concentration for at least one ofthe following: L-2-Aminoadipate, L-2-Aminoadipate 6-semialdehyde, NADP+,NADPH, AMP, ATP, H₂O and/or pyrophosphate.

[0092] wherein a change in concentration for any of the above substancesindicates that said test compound is a candidate for an antibiotic.

[0093] or,

[0094] a) contacting L-2-Aminoadipate 6-semialdehyde, NADP+, AMP,pyrophosphate, and H₂O with an α-Aminoadipate Reductase;

[0095] b) contacting L-2-Aminoadipate 6-semialdehyde, NADP+, AMP,pyrophosphate, and H₂O with an α-Aminoadipate Reductase and said testcompound; and

[0096] c) determining the change in concentration for at least one ofthe following: L-2-Aminoadipate, L-2-Aminoadipate 6-semialdehyde, NADP+,NADPH, AMP, ATP, H₂O and/or pyrophosphate. wherein a change inconcentration for any of the above substances indicates that said testcompound is a candidate for an antibiotic.

[0097] Enzymatically active fragments of a fungal α-AminoadipateReductase are also useful in the methods of the invention. For example,a polypeptide comprising at least 100 consecutive amino acid residues ofa fungal α-Aminoadipate Reductase may be used in the methods of theinvention. In addition, a polypeptide having at least 50%, 60%, 70%,80%, 90%, 95% or at least 98% sequence identity with a fungalα-Aminoadipate Reductase may be used in the methods of the invention.Most preferably, the polypeptide has at least 50% sequence identity witha fungal α-Aminoadipate Reductase and at least 10%, 25%, 75% or at least90% of the activity thereof.

[0098] Thus, the invention provides a method for identifying a testcompound as a candidate for a fungicide, comprising:

[0099] a) contacting L-2-Aminoadipate and NADPH and ATP with apolypeptide selected from the group consisting of: a polypeptide havingat least 50% sequence identity with an α-Aminoadipate Reductase, apolypeptide having at least 50% sequence identity with an α-AminoadipateReductase and having at least 10% of the activity thereof, and apolypeptide comprising at least 100 consecutive amino acids of anα-Aminoadipate Reductase

[0100] b) contacting L-2-Aminoadipate and NADPH and ATP with saidpolypeptide and said test compound; and

[0101] c) determining the change in concentration for at least one ofthe following: L-2-Aminoadipate, L-2-Aminoadipate 6-semialdehyde, NADP+,NADPH, AMP, ATP, H₂O and/or pyrophosphate.

[0102] wherein a change in concentration for any of the above substancesindicates that said test compound is a candidate for an antibiotic.

[0103] or,

[0104] a) contacting L-2-Aminoadipate 6-semialdehyde, NADP+, AMP,pyrophosphate, and H₂O with a polypeptide selected from the groupconsisting of: a polypeptide having at least 50% sequence identity withan α-Aminoadipate Reductase , a polypeptide having at least 50% sequenceidentity with an α-Aminoadipate Reductase and at least 10% of theactivity thereof, and a polypeptide comprising at least 100 consecutiveamino acids of an α-Aminoadipate Reductase

[0105] b) contacting L-2-Aminoadipate 6-semialdehyde, NADP+, AMP,pyrophosphate, and H₂O, with said polypeptide and said test compound;and

[0106] c) determining the change in concentration for at least one ofthe following, L-2-Aminoadipate, L-2-Aminoadipate 6-semialdehyde, NADP+,NADPH, AMP, ATP, H₂O and/or pyrophosphate;

[0107] wherein a change in concentration for any of the above substancesindicates that said test compound is a candidate for an antibiotic.

[0108] For the in vitro enzymatic assays, α-Aminoadipate Reductaseprotein and derivatives thereof may be purified from a fungus or may berecombinantly produced in and purified from an archael, bacterial,fungal, or other eukaryotic cell culture. Preferably these proteins areproduced using an E. coli, yeast, or filamentous fungal expressionsystem. Methods for the purification of α-Aminoadipate Reductase may bedescribed in Jaklitsch and Kubicek (Jaklitsch, W. M. and C. P. Kubicek(1990) Biochem J 269: 247-53 (PMID: 2115771)). Other methods for thepurification of α-Aminoadipate Reductase proteins and polypeptides areknown to those skilled in the art.

[0109] As an alternative to in vitro assays, the invention also providescell based assays. In one embodiment, the invention provides a methodfor identifying a test compound as a candidate for a antibiotic,comprising:

[0110] a) measuring the expression of an α-Aminoadipate Reductase in acell, cells, tissue, or an organism in the absence of said compound;

[0111] b) contacting said cell, cells, tissue, or organism with saidtest compound and measuring the expression of said α-AminoadipateReductase in said cell, cells, tissue, or organism;

[0112] c) comparing the expression of α-Aminoadipate Reductase in steps(a) and (b);

[0113] wherein a lower expression in the presence of said test compoundindicates that said compound is a candidate for an antibiotic.

[0114] Expression of α-Aminoadipate Reductase can be measured bydetecting the AAR1 primary transcript or mRNA, α-Aminoadipate Reductasepolypeptide, or α-Aminoadipate Reductase enzymatic activity. Methods fordetecting the expression of RNA and proteins are known to those skilledin the art. See, for example, Current Protocols in Molecular BiologyAusubel et al., eds., Greene Publishing and Wiley-Interscience, NewYork, 1995. The method of detection is not critical to the invention.Methods for detecting AAR1 RNA include, but are not limited toamplification assays such as quantitative reverse transcriptase-PCR,and/or hybridization assays such as Northern analysis, dot blots, slotblots, in-situ hybridization, transcriptional fusions using an AAR1promoter fused to a reporter gene, DNA assays, and microarray assays.

[0115] Methods for detecting protein expression include, but are notlimited to, immunodetection methods such as Western blots, ELISA assays,polyacrylamide gel electrophoresis, mass spectroscopy, and enzymaticassays. Also, any reporter gene system may be used to detect AAR1protein expression. For detection using gene reporter systems, apolynucleotide encoding a reporter protein is fused in frame with AAR1,so as to produce a chimeric polypeptide. Methods for using reportersystems are known to those skilled in the art.

[0116] Chemicals, compounds or compositions identified by the abovemethods as modulators, preferably inhibitors, of AAR1 expression oractivity can then be used to control fungal growth. Diseases such asrusts, mildews, and blights spread rapidly once established. Fungicidesare thus routinely applied to growing and stored crops as a preventivemeasure, generally as foliar sprays or seed dressings. For example,compounds that inhibit fungal growth can be applied to a fungus orexpressed in a fungus, in order to prevent fungal growth. Thus, theinvention provides a method for inhibiting fungal growth, comprisingcontacting a fungus with a compound identified by the methods of theinvention as having antifungal activity.

[0117] Antifungals and antifungal inhibitor candidates identified by themethods of the invention can be used to control the growth of undesiredfungi, including ascomycota, zygomycota, basidiomycota, chytridiomycota,and lichens.

[0118] Examples of undesired fungi include, but are not limited toPowdery Scab (Spongospora subterranea), Grey Mould (Botrytis cinerea),White Rot (Armillaria mellea), Heartrot Fungus (Ganoderma adspersum),Brown-Rot (Piptoporus betulinus), Corn Smut (Ustilago maydis), Heartrot(Polyporus squamosus), Gray Leaf Spot (Cercospora zeae-maydis), HoneyFungus (Armillaria gallica), Root rot (Armillaria luteobubalina),Shoestring Rot (Armillaria ostoyae), Banana Anthracnose Fungus(Colletotrichum musae), Apple-rotting Fungus (Monilinia fructigena),Apple-rotting Fungus (Penicillium expansum), Clubroot Disease(Plasmodiophora brassicae), Potato Blight (Phytophthora infestans), Rootpathogen (Heterobasidion annosum), Take-all Fungus (Gaeumannomycesgraminis), Dutch Elm Disease (Ophiostoma ulmi), Bean Rust (Uromycesappendiculatus), Northern Leaf Spot (Cochliobolus carbonum), MiloDisease (Periconia circinata), Southern Corn Blight (Cochliobolusheterostrophus), Leaf Spot (Cochliobolus lunata), Brown Stripe(Cochliobolus stenospilus), Panama disease (Fusarium oxysporum), WheatHead Scab Fungus (Fusarium graminearum), Cereal Foot Rot (Fusariumculmorum), Potato Black Scurf (Rhizoctonia solani), Wheat Black StemRust (Puccinia graminis), White mold (Sclerotinia sclerotiorum),diseases of animals such as infections of lungs, blood, brain, skin,scalp, nails or other tissues (Aspergillus fumigatus Aspergillus sp.Fusraium sp., Trichophyton sp., Epidermophyton sp., and Microsporum sp.,and the like).

[0119] Also provided is a method of screening for an antibiotic bydetermining whether a test compound is active against the geneidentified (SEQ ID NO: 1 or SEQ ID NO: 2), its gene product (SEQ ID NO:3), or the biochemical pathway or pathways it functions on.

[0120] In one particular embodiment, the method is performed byproviding an organism having a first form of the gene corresponding toeither SEQ ID NO: 1 or SEQ ID NO: 2, either a normal form, a mutantform, a homologue, or a heterologous AAR1 gene that performs a similarfunction as AAR1. The first form of AAR1 may or may not confer a growthconditional phenotype, i.e., a lysine requiring phenotype, and/or ahypersensitivity or hyposensitivity phenotype on the organism havingthat altered form. In one particular embodiment a mutant form contains atransposon insertion. A comparison organism having a second form of anAAR1, different from the first form of the gene is also provided, andthe two organisms are separately contacted with a test compound. Thegrowth of the two organisms in the presence of the test compound is thencompared.

[0121] Thus, in one embodiment, the invention provides a method foridentifying a test compound as a candidate for an antibiotic,comprising:

[0122] a) providing cells having one form of an α-Aminoadipate Reductasegene, and providing comparison cells having a different form of anα-Aminoadipate Reductase gene,

[0123] b) contacting said cells and said comparison cells with a testcompound and determining the growth of said cells and comparison cellsin the presence of the test compound,

[0124] wherein a difference in growth between said cells and saidcomparison cells in the presence of said test compound indicates thatsaid test compound is a candidate for an antibiotic.

[0125] It is recognized in the art that the optional determination ofthe growth of said first organism and said comparison second organism inthe absence of any test compounds may be performed to control for anyinherent differences in growth as a result of the different genes. It isalso recognized that any combination of two different forms of an AAR1gene, including normal genes, mutant genes, homologues, and functionalhomologues may be used in this method. Growth and/or proliferation of anorganism is measured by methods well known in the art such as opticaldensity measurements, and the like. In a preferred embodiment theorganism is Magnaporthe grisea.

[0126] Conditional lethal mutants may identify particular biochemicaland/or genetic pathways given that at least one identified target geneis present in that pathway. Knowledge of these pathways allows for thescreening of test compounds as candidates for antibiotics as inhibitorsof the substrates, products and enzymes of the pathway. Pathways knownin the art may be found at the Kyoto Encyclopedia of Genes and Genomesand in standard biochemistry texts (Lehninger, A., D. Nelson, et al.(1993) Principles of Biochemistry. New York, Worth Publishers).

[0127] Thus, in one embodiment, the invention provides a method forscreening for test compounds acting against the biochemical and/orgenetic pathway or pathways in which AAR1 functions, comprising:

[0128] a) providing cells having one form of a gene in the lysinebiochemical and/or genetic pathway and providing comparison cells havinga different form of said gene.

[0129] b) contacting said cells and comparison cells with a said testcompound,

[0130] c) determining the growth of said cells and comparison cells inthe presence of said test compound.

[0131] wherein a difference in growth between said cells and saidcomparison cells in the presence of said compound indicates that saidcompound is a candidate for an antibiotic.

[0132] The use of multi-well plates for screening is a format thatreadily accommodates multiple different assays to characterize variouscompounds, concentrations of compounds, and fungal strains in varyingcombinations and formats. Certain testing parameters for the screeningmethod can significantly affect the identification of growth inhibitors,and thus can be manipulated to optimize screening efficiency and/orreliability. Notable among these factors are variable sensitivities ofdifferent mutants, increasing hypersensitivity with increasingly lesspermissive conditions, an apparent increase in hypersensitivity withincreasing compound concentration, and other factors known to those inthe art.

[0133] Conditional lethal mutants may identify particular biochemicaland/or genetic pathways given that at least one identified target geneis present in that pathway. Knowledge of these pathways allows for thescreening of test compounds as candidates for antibiotics. Pathwaysknown in the art may be found at the Kyoto Encyclopedia of Genes andGenomes and in standard biochemistry texts (Lehninger, A., D. Nelson, etal. (1993) Principles of Biochemistry. New York, Worth Publishers).Thus, in one embodiment, the invention provides a method for screeningfor test compounds acting against the biochemical and/or genetic pathwayor pathways in which AAR1 functions, comprising:

[0134] (a) providing paired growth media; comprising a first medium anda second medium, wherein said second medium contains a higher level oflysine than said first medium;

[0135] (b) contacting an organism with said test compound;

[0136] (c) inoculating said first and second media with said organism;and

[0137] (d) determining the growth of said organism;

[0138] wherein a difference in growth of the organism between said firstand second media indicates that said test compound is a candidate for anantibiotic.

[0139] It is recognized in the art that the optional determination ofthe growth of said organism in the paired media in the absence of anytest compounds may be performed to control for any inherent differencesin growth as a result of the different media. Growth and/orproliferation of an organism is measured by methods well known in theart such as optical density measurements, and the like. In a preferredembodiment, the organism is Magnaporthe grisea.

EXPERIMENTAL Example 1

[0140] Construction of Plasmids with a Transposon Containing aSelectable Marker.

[0141] Construction of Sif transposon: Sif was constructed using theGPS3 vector from the GPS-M mutagenesis system from New England Biolabs,Inc. (Beverly, Mass.) as a backbone. This system is based on thebacterial transposon Tn7. The following manipulations were done to GPS3according to Sambrook et al. (1989) Molecular Cloning, a LaboratoryManual, Cold Spring Harbor Laboratory Press. The kanamycin resistancegene (npt) contained between the Tn7 arms was removed by EcoRVdigestion. The bacterial hygromycin B phosphotransferase (hph) gene(Gritz and Davies (1983) Gene 25: 179-88 (PMID: 6319235)) under controlof the Aspergillus nidulans trpC promoter and terminator (Mullaney etal. (1985) Mol Gen Genet 199: 37-45 (PMID: 3158796)) was cloned by aHpaI/EcoRV blunt ligation into the Tn7 arms of the GPS3 vector yieldingpSif1. Excision of the ampicillin resistance gene (bla) from pSif1 wasachieved by cutting pSif1 with XmnI and BglI followed by a T4 DNApolymerase treatment to remove the 3′ overhangs left by the BglIdigestion and religation of the plasmid to yield pSif. Top 10F′electrocompetent E. coli cells (Invitrogen) were transformed withligation mixture according to manufacturer's recommendations.Transformants containing the Sif transposon were selected on LB agar(Sambrook et al. (1989) Molecular Cloning, a Laboratory Manual, ColdSpring Harbor Laboratory Press.) containing 50ug/ml of hygromycin B(Sigma Chem. Co., St. Louis, Mo.).

Example 2

[0142] Construction of a Cosmid Library Containing Fungal Genes and aSelectable Marker

[0143] Cosmid libraries were constructed in the pcosKA5 vector (Hamer etal. (2001) Proc Natl Acad Sci USA 98: 5110-15 (PMID: 11296265)) asdescribed in Sambrook et al. (1989) Molecular Cloning, a LaboratoryManual, Cold Spring Harbor Laboratory Press. Cosmid libraries werequality checked by pulsed-field gel electrophoresis, restrictiondigestion analysis, and PCR identification of single genes.

Example 3

[0144] Construction of Cosmids with Transposon Inserted into FungalGenes.

[0145] Sif Transposition into a Cosmid: Transposition of Sif into thecosmid framework was carried out as described by the GPS-M mutagenesissystem (New England Biolabs, Inc.). Briefly, 2 ul of the 10×GPS buffer,70 ng of supercoiled pSIF, 8-12 μg of target cosmid DNA were mixed andtaken to a final volume of 20 ul with water. 1 ul of transposase(TnsABC) was added to the reaction and incubated for 10 minutes at 37°C. to allow the assembly reaction to happen. After the assembly reaction1 ul of start solution was added to the tube, mixed well and incubatedfor 1 hour at 37° C. followed by heat inactivation of the proteins at75° C. for 10 min. Destruction of the remaining untransposed pSif wasdone by PISceI digestion at 37° C. for 2 hours followed by 10 minincubation at 75° C. to inactivate the proteins. Transformation ofTop10F′ electrocompetent cells (Invitrogen) was done according tomanufacturers recommendations. Sif-containing cosmid transformants wereselected by growth on LB agar plates containing 50 ug/ml of hygromycin B(Sigma Chem. Co.) and 100 ug/ml of Ampicillin (Sigma Chem. Co.).

Example 4

[0146] High Throughput Preparation and Verification of Insertion ofTransposon into Fungal Genes

[0147]E. coli strains containing cosmids with transposon insertions werepicked to 96 well growth blocks (Beckman Co.) containing 1.5 ml of TB(Terrific Broth, Sambrook et al. (1989) Molecular Cloning, a LaboratoryManual, Cold Spring Harbor Laboratory Press) supplemented with 50 ug/mlof ampicillin. Blocks were incubated with shaking at 37 C overnight. E.coli cells were pelleted by centrifugation and cosmids were isolated bya modified alkaline lysis method (Marra et al. (1997) Genome Res 7:1072-84 (PMID: 9371743)). DNA quality was checked by electrophoresis onagarose gels. Cosmids were sequenced using primers from the ends of eachtransposon and commercial dideoxy sequencing kits (Big Dye Terminators,Perkin Elmer Co.). Sequencing reactions were analyzed on an ABI377 DNAsequencer (Perkin Elmer Co.).

[0148] DNA sequences adjacent to the site of the insertion werecollected and used to search DNA and protein databases using the BLASTalgorithms (Altschul et al. (1997) Nucleic Acids Res 25: 3389-3402(PMID: 9254694)). A single insertion of SIF into the Magnaporthe griseaAAR1 gene was chosen for further analysis. This construct was designatedcpgmra0015019g12 and it contains the SIF transposon approximatelybetween amino acids 678 and 679 relative to the Neurospora crassahomologue (total length-1174 amino acids, GENBANK: 9367248 of accessionnumber AL389890).

Example 5

[0149] Preparation of Cosmid DNA and Transformation of the FungusMagnaporthe grisea

[0150] Cosmid DNA from the AAR1 transposon tagged cosmid clone wasprepared using QIAGEN Plasmid Maxi Kit (QIAGEN), and digested by PI-PspI(New England Biolabs, Inc.). Fungal electro-transformation was performedessentially as described (Wu et al. (1997) MPMI 10: 700-708). Briefly,M. grisea strain Guy 11 was grown in complete liquid media (Talbot etal. (1993) Plant Cell 5: 1575-1590 (PMID: 8312740)) shaking at 120 rpmfor 3 days at 25° C. in the dark. Mycelia was harvested and washed withsterile H₂O and digested with 4 mg/ml beta-glucanase (InterSpex) for 4-6hours to generate protoplasts. Protoplasts were collected bycentrifugation and resuspended in 20% sucrose at the concentration of2×10⁸ protoplasts/ml. 50 ul protoplast suspension was mixed with 10-20ug of the cosmid DNA and pulsed using Gene Pulser II (BioRad) set withthe following parameters: resistance 200 ohm, capacitance 25 uF, voltage0.6 kV. Transformed protoplasts were regenerated in complete agar media(CM, Talbot et al. (1993) Plant Cell 5: 1575-1590 (PMID: 8312740)) withthe addition of 20% sucrose for one day, then overlayed with CM agarmedia containing hygromycin B (250 ug/ml) to select transformants.Transformants were screened for homologous recombination events in thetarget gene by PCR (Hamer et al. (2001) Proc Natl Acad Sci USA 98:5110-15 (PMID: 11296265)). Two independent strains were identified andare hereby referred to as KO1-1 and KO1-11, respectively.

Example 6

[0151] Effect of Transposon Insertion on Magnaporthe Pathogenicity

[0152] The target fungal strains, KO1-1 and KO1-11, obtained in Example5 and the wild type strain, Guy11, were subjected to a pathogenicityassay to observe infection over a 1-week period. Rice infection assayswere performed using Indian rice cultivar CO39 essentially as describedin Valent et al. ((1991) Genetics 127: 87-101 (PMID: 2016048)). Allthree strains were grown for spore production on complete agar media.Spores were harvested and the concentration of spores adjusted for wholeplant inoculations. Two-week-old seedlings of cultivar CO39 were sprayedwith 12 ml of conidial suspension (5×10⁴conidia per ml in 0.01% Tween-20(Polyoxyethylensorbitan monolaureate) solution). The inoculated plantswere incubated in a dew chamber at 27° C. in the dark for 36 hours, andtransferred to a growth chamber (27° C. 12 hours/21° C. 12 hours 70%humidity) for an additional 5.5 days. Leaf samples were taken at 3, 5,and 7 days post-inoculation and examined for signs of successfulinfection (i.e. lesions). FIG. 2 shows the effects of AAR1 genedisruption on Magnaporthe infection at five days post-inoculation.

Example 7

[0153] Verification of Gene Function by Analysis of NutritionalRequirements

[0154] The fungal strains, KO1-1 and KO1-11, containing the AAR1disrupted gene obtained in Example 5 were analyzed for their nutritionalrequirement for lysine using the PM5 phenotype microarray from Biolog,Inc. (Hayward, Calif.). The PM5 plate tests for the auxotrophicrequirement for 94 different metabolites. The inoculating fluid consistsof 0.05% Phytagel, 0.03% Pluronic F68, 1% glucose, 23.5 mM NaNO₃, 6.7 mMKCl, 3.5 mM Na₂SO₄, 11 mM KH₂PO₄, 0.01%p-iodonitrotetrazolium violet,0.1 mM MgCl₂, 1.0 mM CaCl₂ and trace elements. Final concentrations oftrace elements are: 7.6 uM ZnCl₂, 2.5 μM MnCl₂.4H₂O, 1.8 μM FeCl₂.4H₂O,0.71 μM CoCl₂.6H₂O, 64 μM CuCl₂.2H₂O, 0.62 μM Na₂MoO₄, 18 μM H₃BO₃. pHadjusted to 6.0 with NaOH. Spores for each strain were harvested intothe inoculating fluid. The spore concentrations were adjusted to 2×10⁵spores/ml. 100 μl of spore suspension were deposited into each well ofthe microtiter plates. The plates were incubated at 25° C. for 7 days.Optical density (OD) measurements at 490 nm and 750 nm were taken daily.The OD₄₉₀ measures the extent of tetrazolium dye reduction and the levelof growth, and OD₇₅₀ measures growth only. Turbidity=OD₄₉₀+OD₇₅₀. Dataconfirming the annotated gene function is presented as a graph ofTurbidity vs. Time showing both the mutant fungi and the wild-typecontrol in the absence (FIG. 3A) and presence (FIG. 3B) of L-lysine.

Example 8

[0155] Cloning and Expression Strategies, Extraction and Purification ofα-Aminoadipate Reductase Protein.

[0156] The following protocol may be employed to obtain a purifiedα-Aminoadipate Reductase protein.

[0157] Cloning and expression strategies:

[0158] An AAR1 cDNA gene can be cloned into E. coli (pETvectors-Novagen), Baculovirus (Pharmingen) and Yeast (Invitrogen)expression vectors containing His/fusion protein tags. Evaluate theexpression of recombinant protein by SDS-PAGE and Western blot analysis.

[0159] Extraction:

[0160] Extract recombinant protein from 250 ml cell pellet in 3 ml ofextraction buffer By sonicating 6 times, with 6 sec pulses at 4° C.Centrifuge extract at 15000×g for 10 min and collect supernatant. Assessbiological activity of the recombinant protein by activity assay.

[0161] Purification:

[0162] Purify recombinant protein by Ni-NTA affinity chromatography(Qiagen).

[0163] Purification protocol: perform all steps at 4° C.:

[0164] Use 3 ml Ni-beads (Qiagen)

[0165] Equilibrate column with the buffer

[0166] Load protein extract

[0167] Wash with the equilibration buffer

[0168] Elute bound protein with 0.5 M imidazole

Example 9

[0169] Assays for Testing Binding of Test Compounds to α-AminoadipateReductase

[0170] The following protocol may be employed to identify test compoundsthat bind to the α-Aminoadipate Reductase protein.

[0171] Purified full-length α-Aminoadipate Reductase polypeptide with aHis/fusion protein tag (Example 8) is bound to a HisGrab™ Nickel CoatedPlate (Pierce, Rockford, Ill.) following manufacturer's instructions.

[0172] Buffer conditions are optimized (e.g. ionic strength or pH,Jaklitsch, W. M. and C. P. Kubicek (1990) Biochem J 269: 247-53 (PMID:2115771)) for binding of radiolabeled ¹⁴C-α-Aminoadipic acid(Sigma-Aldrich Co.) to the bound AAR 1.

[0173] Screening of test compounds is performed by adding test compoundand ¹⁴C-α-Aminoadipic acid (Sigma-Aldrich Co.) to the wells of theHisGrab™ plate containing bound AAR1.

[0174] The wells are washed to remove excess labeled ligand andscintillation fluid (Scintiverse®, Fisher Scientific) is added to eachwell.

[0175] The plates are read in a microplate scintillation counter.

[0176] Candidate compounds are identified as wells with lowerradioactivity as compared to control wells with no test compound added.

[0177] Additionally, a purified polypeptide comprising 10-50 amino acidsfrom the M. grisea α-Aminoadipate Reductase is screened in the same way.A polypeptide comprising 10-50 amino acids is generated by subcloning aportion of the AAR1 gene into a protein expression vector that adds aHis-Tag when expressed (see Example 8). Oligonucleotide primers aredesigned to amplify a portion of the AAR1 gene using the polymerasechain reaction amplification method. The DNA fragment encoding apolypeptide of 10-50 amino acids is cloned into an expression vector,expressed in a host organism and purified as described in Example 8above.

[0178] Test compounds that bind AAR1 are further tested for antibioticactivity. M. grisea is grown as described for spore production onoatmeal agar media (Talbot et al. (1993) Plant Cell 5: 1575-1590 (PMID:8312740)). Spores are harvested into minimal media (Talbot et al. (1993)Plant Cell 5: 1575-1590 (PMID: 8312740)) to a concentration of 2×10⁵spores/ml and the culture is divided. The test compound is added to oneculture to a final concentration of 20-100 μg/ml. Solvent only is addedto the second culture. The plates are incubated at 25° C. for seven daysand optical density measurements at 590 nm are taken daily. The growthcurves of the solvent control sample and the test compound sample arecompared. A test compound is an antibiotic candidate if the growth ofthe culture containing the test compound is less than the growth of thecontrol culture.

Example 10

[0179] Assays for Testing Inhibitors or Candidates for Inhibition ofα-Aminoadipate Reductase Activity

[0180] The enzymatic activity of α-Aminoadipate Reductase is determinedin the presence and absence of candidate compounds in a suitablereaction mixture, such as described by Gray and Bhattacharjee (Gray, GSand Bhattacharjee, JK (1976) Can J Microbiol 22: 1664-7 (PMID: 10066)),or Jaklitsch, W. M. and C. P. Kubicek (1990) Biochem J 269: 247-53(PMID: 2115771). Candidate compounds are identified when a decrease inproducts or a lack of decrease in substrates is detected with thereaction proceeding in either direction.

[0181] Additionally, the enzymatic activity of a polypeptide comprising10-50 amino acids from the M. grisea α-Aminoadipate Reductase isdetermined in the presence and absence of candidate compounds in asuitable reaction mixture, such as described by Gray and Bhattacharjee(Gray, GS and Bhattacharjee, JK (1976) Can J Microbiol 22: 1664-7 (PMID:10066)), or Jaklitsch, W. M. and C. P. Kubicek (1990) Biochem J 269:247-53 (PMID: 2115771). A polypeptide comprising 10-50 amino acids isgenerated by subcloning a portion of the AAR1 gene into a proteinexpression vector that adds a His-Tag when expressed (see Example 8).Oligonucleotide primers are designed to amplify a portion of the AAR1gene using polymerase chain reaction amplification method. The DNAfragment encoding a polypeptide of 10-50 amino acids is cloned into anexpression vector, expressed and purified as described in Example 8above.

[0182] Test compounds identified as inhibitors of AAR1 activity arefurther tested for antibiotic activity. Magnaporthe grisea fungal cellsare grown under standard fungal growth conditions that are well knownand described in the art. M. grisea is grown as described for sporeproduction on oatmeal agar media (Talbot et a. (1993) Plant Cell 5:1575-1590 (PMID: 8312740)). Spores are harvested into minimal media(Talbot et al. (1993) Plant Cell 5: 1575-1590 (PMID: 8312740)) to aconcentration of 2×10⁵ spores/ml and the culture is divided. The testcompound is added to one culture to a final concentration of 20-100μg/ml. Solvent only is added to the second culture. The plates areincubated at 25° C. for seven days and optical density measurements at590 nm are taken daily. The growth curves of the solvent control sampleand the test compound sample are compared. A test compound is anantibiotic candidate if the growth of the culture containing the testcompound is less than the growth of the control culture.

Example 11

[0183] Assays for Testing Compounds or Candidates for Compounds ThatAlter the Expression of an α-Aminoadipate Reductase Gene

[0184]Magnaporthe grisea fungal cells are grown under standard fungalgrowth conditions that are well known and described in the art.Wild-type M. grisea spores are harvested from cultures grown on completeagar or oatmeal agar media after growth for 10-13 days in the light at25° C. using a moistened cotton swab. The concentration of spores isdetermined using a hemacytometer and spore suspensions are prepared in aminimal growth medium to a concentration of 2×10⁵ spores per ml. 25 mlcultures are prepared to which test compounds will be added at variousconcentrations. A culture with no test compound present is included as acontrol. The cultures are incubated at 25° C. for 3 days after whichtest compound or solvent only control is added. The cultures areincubated an additional 18 hours. Fungal mycelia is harvested byfiltration through Miracloth (CalBiochem®, La Jolla, Calif.), washedwith water and frozen in liquid nitrogen. Total RNA is extracted withTRIZOL® Reagent using the methods provided by the manufacturer (LifeTechnologies, Rockville, Md.). Expression is analyzed by Northernanalysis of the RNA samples as described (Sambrook et al. (1989)Molecular Cloning, a Laboratory Manual, Cold Spring Harbor LaboratoryPress) using a radiolabeled fragment of the AAR1 gene as a probe. Testcompounds resulting in a reduced level of AAR1 mRNA relative to theuntreated control sample are identified as candidate antibioticcompounds.

Example 12

[0185] In Vivo Cell Based Assay Screening Protocol with a Fungal StrainContaining a Mutant Form of α-Aminoadipate Reductase with No Activity

[0186]Magnaporthe grisea fungal cells containing a mutant form of theAAR1 gene which abolishes enzyme activity, such as a gene containing atransposon insertion (see Examples 4 and 5), are grown under standardfungal growth conditions that are well known and described in the art.Magnaporthe grisea spores are harvested from cultures grown on completeagar medium containing 4 mM L-lysine (Sigma-Aldrich Co.) after growthfor 10-13 days in the light at 25° C. using a moistened cotton swab. Theconcentration of spores is determined using a hemacytometer and sporesuspensions are prepared in a minimal growth medium containing 100 μML-lysine to a concentration of 2×10⁵ spores per ml. Approximately 4×10⁴spores are added to each well of 96-well plates to which a test compoundis added (at varying concentrations). The total volume in each well is200 μl. Wells with no test compound present (growth control), and wellswithout cells are included as controls (negative control). The platesare incubated at 25° C. for seven days and optical density measurementsat 590 nm are taken daily. Wild type cells are screened under the sameconditions. The effect of each compound on the mutant and wild-typefungal strains is measured against the growth control and the percent ofinhibition is calculated as the OD₅₉₀ (fungal strain plus testcompound)/OD₅₉₀ (growth control)×100. The percent of growth inhibitionas a result of a test compound on a fungal strain and that on the wildtype cells are compared. Compounds that show differential growthinhibition between the mutant and the wild type are identified aspotential antifungal compounds. Similar protocols may be found in Kirschand DiDomenico ((1994) Biotechnology 26: 177-221 (PMID: 7749303)).

Example 13

[0187] In Vivo Cell Based Assay Screening Protocol with a Fungal StrainContaining a Mutant Form of α-Aminoadipate Reductase with ReducedActivity

[0188]Magnaporthe grisea fungal cells containing a mutant form of theAAR1 gene, such as a promoter truncation that reduces expression, aregrown under standard fungal growth conditions that are well known anddescribed in the art. A promoter truncation is made by deleting aportion of the promoter upstream of the transcription start site usingstandard molecular biology techniques that are well known and describedin the art (Sambrook et al. (1989) Molecular Cloning, a LaboratoryManual, Cold Spring Harbor Laboratory Press). Magnaporthe grisea sporesare harvested from cultures grown on complete agar medium containing 4mM L-lysine (Sigma-Aldrich Co.) after growth for 10-13 days in the lightat 25° C. using a moistened cotton swab. The concentration of spores isdetermined using a hemacytometer and spore suspensions are prepared in aminimal growth medium to a concentration of 2×10⁵ spores per ml.Approximately 4×10⁴ spores are added to each well of 96-well plates towhich a test compound is added (at varying concentrations). The totalvolume in each well is 200 μl. Wells with no test compound present(growth control), and wells without cells are included as controls(negative control). The plates are incubated at 25° C. for seven daysand optical density measurements at 590 nm are taken daily. Wild typecells are screened under the same conditions. The effect of eachcompound on the mutant and wild-type fungal strains is measured againstthe growth control and the percent of inhibition is calculated as theOD₅₉₀ (fungal strain plus test compound)/OD₅₉₀ (growth control)×100. Thepercent of growth inhibition as a result of a test compound on a fungalstrain and that on the wild-type cells are compared. Compounds that showdifferential growth inhibition between the mutant and the wild type areidentified as potential antifungal compounds. Similar protocols may befound in Kirsch and DiDomenico ((1994) Biotechnology 26: 177-221 (PMID:7749303)).

Example 14

[0189] In Vivo Cell Based Assay Screening Protocol with a Fungal StrainContaining a Mutant Form of a Lysine Biosynthetic Gene with No Activity

[0190]Magnaporthe grisea fungal cells containing a mutant form of a genein the lysine biosynthetic pathway (e.g. HCS1 (E.C. 4.1.3.21)) are grownunder standard fungal growth conditions that are well known anddescribed in the art. Magnaporthe grisea spores are harvested fromcultures grown on complete agar medium containing 4 mM L-lysine(Sigma-Aldrich Co.) after growth for 10-13 days in the light at 25° C.using a moistened cotton swab. The concentration of spores is determinedusing a hemacytometer and spore suspensions are prepared in a minimalgrowth medium containing 100 μM L-lysine to a concentration of 2×10⁵spores per ml. Approximately 4×10⁴ spores or cells are harvested andadded to each well of 96-well plates to which growth media is added inaddition to an amount of test compound (at varying concentrations). Thetotal volume in each well is 200 μl. Wells with no test compoundpresent, and wells without cells are included as controls. The platesare incubated at 25° C. for seven days and optical density measurementsat 590 nm are taken daily. Wild type cells are screened under the sameconditions. The effect of each compound on the mutant and wild-typefungal strains is measured against the growth control and the percent ofinhibition is calculated as the OD₅₉₀ (fungal strain plus testcompound)/OD₅₉₀ (growth control)×100. The percent of growth inhibitionas a result of a test compound on a fungal strain and that on the wildtype cells are compared. Compounds that show differential growthinhibition between the mutant and the wild-type are identified aspotential antifungal compounds. Similar protocols may be found in Kirschand DiDomenico ((1994) Biotechnology 26: 177-221 (PMID: 7749303)).

Example 15

[0191] In Vivo Cell Based Assay Screening Protocol with a Fungal StrainContaining a Mutant Form of a Lysine Biosynthetic Gene with ReducedActivity

[0192]Magnaporthe grisea fungal cells containing a mutant form of a genein the lysine biosynthetic pathway (e.g. HCS1 (E.C. 4.1.3.21)), such asa promoter truncation that reduces expression, are grown under standardfungal growth conditions that are well known and described in the art. Apromoter truncation is made by deleting a portion of the promoterupstream of the transcription start site using standard molecularbiology techniques that are well known and described in the art(Sambrook et al. (1989) Molecular Cloning, a Laboratory Manual, ColdSpring Harbor Laboratory Press). Magnaporthe grisea fungal cellscontaining a mutant form of are grown under standard fungal growthconditions that are well known and described in the art. Magnaporthegrisea spores are harvested from cultures grown on complete agar mediumcontaining 4 mM L-lysine (Sigma-Aldrich Co.) after growth for 10-13 daysin the light at 25° C. using a moistened cotton swab. The concentrationof spores is determined using a hemacytometer and spore suspensions areprepared in a minimal growth medium to a concentration of 2×10⁵ sporesper ml. Approximately 4×10⁴ spores or cells are harvested and added toeach well of 96-well plates to which growth media is added in additionto an amount of test compound (at varying concentrations). The totalvolume in each well is 200 μl. Wells with no test compound present, andwells without cells are included as controls. The plates are incubatedat 25° C. for seven days and optical density measurements at 590 nm aretaken daily. Wild type cells are screened under the same conditions. Theeffect of each compound on the mutant and wild-type fungal strains ismeasured against the growth control and the percent of inhibition iscalculated as the OD₅₉₀ (fungal strain plus test compound)/OD₅₉₀ (growthcontrol)×100. The percent of growth inhibition as a result of a testcompound on a fungal strain and that on the wild type cells arecompared. Compounds that show differential growth inhibition between themutant and the wild type are identified as potential antifungalcompounds. Similar protocols may be found in Kirsch and DiDomenico((1994) Biotechnology 26: 177-221 (PMID: 7749303)).

Example 16

[0193] In Vivo Cell Based Assay Screening Protocol with a Fungal StrainContaining a Fungal AAR1 and a Second Fungal Strain Containing aHeterologous AAR1 Gene

[0194] Wild-type Magnaporthe grisea fungal cells and M. grisea fungalcells lacking a functional AAR1 gene and containing an AAR1 gene fromPenicillium chrysogenum (Genbank accession Y1 3967, 56% sequenceidentity) are grown under standard fungal growth conditions that arewell known and described in the art. A M. grisea strain carrying aheterologous AAR1 gene is made as follows:

[0195] A M. grisea strain is made with a nonfunctional AAR1 gene, suchas one containing a transposon insertion in the native gene (seeExamples 4 and 5).

[0196] A construct containing a heterologous AAR1 gene is made bycloning the AAR1 gene from Penicillium chrysogenum into a fungalexpression vector containing a trpC promoter and terminator (e.g.pCB1003, Carroll et al. (1994) Fungal Gen News Lett 41: 22) usingstandard molecular biology techniques that are well known and describedin the art (Sambrook et al. (1989) Molecular Cloning, a LaboratoryManual, Cold Spring Harbor Laboratory Press).

[0197] The said construct is used to transform the M. grisea strainlacking a functional AAR1 gene (see Example 5). Transformants areselected on minimal agar medium lacking L-lysine. Only transformantscarrying a functional AAR1 gene will grow.

[0198] Wild-type strains of Magnaporthe grisea and strains containing aheterologous form of AAR1 are grown under standard fungal growthconditions that are well known and described in the art. Magnaporthegrisea spores are harvested from cultures grown on complete agar mediumafter growth for 10-13 days in the light at 25° C. using a moistenedcotton swab. The concentration of spores is determined using ahemacytometer and spore suspensions are prepared in a minimal growthmedium to a concentration of 2×10⁵ spores per ml. Approximately 4×10⁴spores or cells are harvested and added to each well of 96-well platesto which growth media is added in addition to an amount of test compound(at varying concentrations). The total volume in each well is 200 μl.Wells with no test compound present, and wells without cells areincluded as controls. The plates are incubated at 25° C. for seven daysand optical density measurements at 590nm are taken daily. The effect ofeach compound on the wild-type and heterologous fungal strains ismeasured against the growth control and the percent of inhibition iscalculated as the OD₅₉₀ (fungal strain plus test compound)/OD₅₉₀ (growthcontrol)×100. The percent of growth inhibition as a result of a testcompound on the wild-type and heterologous fungal strains are compared.Compounds that show differential growth inhibition between the wild-typeand heterologous strains are identified as potential antifungalcompounds with specificity to the native or heterologous AAR1 geneproducts. Similar protocols may be found in Kirsch and DiDomenico((1994) Biotechnology 26: 177-221 (PMID: 7749303)).

Example 17

[0199] Pathway Specific In Vivo Assay Screening Protocol

[0200]Magnaporthe grisea fungal cells are grown under standard fungalgrowth conditions that are well known and described in the art.Wild-type M. grisea spores are harvested from cultures grown on oatmealagar media after growth for 10-13 days in the light at 25° C. using amoistened cotton swab. The concentration of spores is determined using ahemocytometer and spore suspensions are prepared in a minimal growthmedium and a minimal growth medium containing 4 mM L-lysine(Sigma-Aldrich Co.) to a concentration of 2×10⁵ spores per ml. Theminimal growth media contains carbon, nitrogen, phosphate, and sulfatesources, and magnesium, calcium, and trace elements (for example, seeinoculating fluid in Example 7). Spore suspensions are added to eachwell of a 96-well microtiter plate (approximately 4×10⁴ spores/well).For each well containing a spore suspension in minimal media, anadditional well is present containing a spore suspension in minimalmedium containing 4 mM L-lysine. Test compounds are added to wellscontaining spores in minimal media and minimal media containingL-lysine. The total volume in each well is 200 μl. Both minimal mediaand L-lysine containing media wells with no test compound are providedas controls. The plates are incubated at 25° C. for seven days andoptical density measurements at 590 nm are taken daily. A compound isidentified as a candidate for an antibiotic acting against the lysinebiosynthetic pathway when the observed growth in the well containingminimal media is less than the observed growth in the well containingL-lysine as a result of the addition of the test compound. Similarprotocols may be found in Kirsch and DiDomenico ((1994) Biotechnology26: 177-221 (PMID: 7749303)).

[0201] While the foregoing describes certain embodiments of theinvention, it will be understood by those skilled in the art thatvariations and modifications may be made and still fall within the scopeof the invention. The foregoing examples are intended to exemplifyvarious specific embodiments of the invention and do not limit its scopein any manner.

1 3 1 3567 DNA Magnaporthe grisea 1 atggcgcatc agctccccga cccaacggtcgacctcgact ggtctggcta cgtcggcgcc 60 attcatgaga tctttgccac taatgcccagaagcacccgg agcgggtgtg cgtgatcgag 120 acagagtcct ccgaggcacc ggaaaggatatttacctaca agcagatctt tgaggcgtca 180 aatgtcctgg cgcactacct acatgatgctggagtcacta atggcgatgt ggtcatgatc 240 tgggcgcata ggtcagttga cctggttgtcagcatcatgg gtgttcttgc tgccggagct 300 acattcagtg tccttgaccc attatacccgccatctcgtc agcagatcta cctcgaagta 360 tccggcccga ccgcccttgt acaaatcgcgcgcgccaccg acgaggccgg cccgttggcc 420 cccctcgtgc gcaggtacat cgacgaggagctgaagctga aggccgaggt tccgtcacta 480 cgcatcggcg acgatggcca cctctcgggtggagagatca acggcgctga tgtttttgcc 540 agcgtgcgcg gcaaggcatc ctcaccgcctgcagacattg aggtcggacc cgactcgaac 600 cccacactta gcttcacgtc aggctcggaaggccggccta agggcgtgct tggtcgacac 660 tacagcttgg ccaagtattt tcgatggatggccgagacgt tcggcatggg cgaagagagc 720 cgcttcacac tgctctcggg tatcgcgcacgaccctgtgc agcgagatat cttcacgcca 780 ctgtacctgg gcgcgcgcct actggtgccgtccaaggaga atattgcaca cgagcgtcta 840 gcagagtggt tcaagcgctt tgaaccaacagtgacacacc tgacgccggc catgggtcag 900 attttagtcg gcggcgctac cgcacagttccctgccctga aaacagccta cttcgtcggc 960 gatgtgctga cgacgcgaga ctgccgcagtctgcgtgagc tcgcggcaaa cgttgacatt 1020 gttaacatgt acgggacgac tgagacgagcagagctgtca gctactacaa gatcccgaac 1080 cgcgcctcag acccggactt tctggagagattgggcaagg acacaatccc tgctggaact 1140 ggcatggaaa acgttcagct tttggttgtcaaccgggaag ataggacaaa gctttgtggt 1200 atcggcgaag tgggcgagat ctacgtgcgtgcggctggtc tggctgaggg ctacaagggc 1260 gaccccgctt tgaacgaaca gaagttcctgatgaactggt tcgtggataa caacaagtgg 1320 gttgaggcgg acagggtgca cccaaccaaggatgcggcat ggagaaagta ctacaagggc 1380 cctctcgaca ggctgtaccg cacaggtgacctcggaaagt atttggattc gggcgatgtg 1440 gaatgcactg gtcgtgcaga cgatcaagtcaaaatcaggg gcttcaggat tgaactcaac 1500 gacattgaca gcaacctgag tcagagctccctcatcaggg actgcaagac gcttgtgcga 1560 agggacaaga acgaggagcc gaagctggtcagctacgttg tgcctgagct caagcaatgg 1620 ccccaatggc tcaagcttca tggctacgaggatgctgaag acgatgaggg cacgcaaatt 1680 ggagcaacca aggtatactt caagaggtatcgtcgtatgc aggctgagct tcgcgaccat 1740 ctcaagtcaa ggttgccgac ctacgccgttcctagcatct tcattgtcct ggagaagctg 1800 ccattaaacc ccaacggcaa ggttgacaagcccaatctac cttttcccga tattgccgag 1860 caaaccgcgg aagcttcaag cgaggagattgagcgatggg agtctttgac cgagactgag 1920 cgtgctgttg ccaccaggtg ggctgcgttgatccagggtc tgaacgaaaa gtcgatcgcg 1980 cccgataacc acttctttga cctcggcggtcacagtattc tggcacagca aatgctgctc 2040 gacgtgcgca agcagatggg tgctaatgtgtctatcaaca ccctttacga gaaccccacg 2100 cttggggcat tcagtcttca gattgacaagcatcttggag cagccaatga tgctagcacc 2160 agccaagtcg aggatgaggc aaactcgtatgccaaggctc gtgatgatct cgttaagaaa 2220 cttccagcct catacaagac agcagatccgtcgtcgatcc gggcgtcatc cagacctacc 2280 atcttcctga ctggcgcaac gggtttcttgggtgcctttt tgatccgcga tatcctgcag 2340 aggacgagcc gacagctgaa gctcatcgcacacgtgcgtg ccaaggacca aaaagcggcg 2400 acagagcgtc tgacgcggtc actacagggctacggtatct ggcgcgacga gtgggctggg 2460 cgcctttcct gcgtagtggg tgacctagccaagccgcaac ttggtattga tcagcccaca 2520 tgggagcgcc tgtcgaacga ggtggacgtagttatccaca acggtgcgac agtccactgg 2580 gtgcgccgct ggcaagacat gctggccgccaacgtcacat cgacaatcga ggccatgcgg 2640 ctgtgcaacg agggcaagcc aaagttgttcactttcgtca gctcaactag cgtcttggac 2700 actgagcact atgtgcagtt gtcggagaggcaactgagca ccggccaagg cgccgtcccc 2760 gagtctgacg acctcgaagg cagtgccactggcttgggta caggttacgg ccaaaccaag 2820 tggatctcgg agcagctcgt cagggaggcgggccgacgcg gacttcgagg ctccgttgtc 2880 aggccaggct acattctggg agatttcgagtcgggatgtt ccaacacaga cgacttcctc 2940 attcgcttcc tcaagggctg tatccagctcggcacgaggc cccgcattct caatactgtc 3000 aatgccgtgc ccgtcaacca cgttgcgcgtgttgttgtcg cggctggtct caaccctgta 3060 cccgtccagg gtaatgaagg tgtccacgtggtccacgtta cgggccaccc gcgcctgcgg 3120 atgaacgagt atctctcgtt gcttgagttctacggctaca aggtgcccga ggtgccgtat 3180 gattcatgga aggaggagct ggagcagtacgtgtctgcgg gcgcgggtgt cgagcgcgac 3240 caggagcagc acgcgctgct acctctcttccacctctgta tctcggacct gcccgccaac 3300 actaaggcac ctgagcttga ggaccaaaacgctgtcgcgg tcctcaaggc ggacgctgag 3360 gcctggaccg ggctcgacga gagcgcgggctacggcatcg gcagggagga cgtcggaagg 3420 tacctccgct acctagccat gatcaagtttgtcccctggc ctacgtcgag gggcaggcct 3480 ttgcctgagg tcagcatcag cacggagcaggtggctgcta tgggtgcagg cgtcggtgga 3540 cgtggtggtg cgggtgcggg acagtga 35672 7586 DNA Magnaporthe grisea 2 catcacgagt cagtttagga agcctggctctgggagagct cccgcgccaa attgccccag 60 aaattaagca tccatgccaa gagcggcttcccgatgtcgc aataccgaat gtcattgtgg 120 atcaattttt tttacattct gcagggatctaattgagctt tgaaaagtcg agtcaccgtc 180 accagagtta ctcactcttc ttttttttcagcaagcatct tggtgggctg ctggcttctt 240 gttttgcttg atgtctgtaa aagattcaatcgaggccagg ggcagtttta agtacatata 300 aactttgcaa aaagctaggg ttcacataccagtcaacgca tacacactgg cgcagtagta 360 ccccgccaat tgtcccccat cacgataaatcgtcaccatg gcgcatcagc tccccgaccc 420 aacggtcgac ctcgactggt ctggctacgtcggcgccatt catgagatct ttgccactaa 480 tgcccagaag cacccggagc gggtgtgcgtgatcgagaca gagtcctccg aggcaccgga 540 aaggatattt acctacaagc agatctttgaggcgtcaaat gtcctggcgc actacctaca 600 tgatgctgga gtcactaatg gcgatgtggtcatgatctgg gcgcataggt cagttgacct 660 ggttgtcagc atcatgggtg ttcttgtaagctgcatcttc cctccctatg aacttgaccg 720 attccccgcc ttgtcttagc cagcactgtttctccgagag aacggaaaca ctgacatgac 780 caaaaccatt cctaggctgc cggagctacattcagtgtcc ttgacccatt atacccgcca 840 tctcgtcagc agatctacct cgaagtatccggcccgaccg cccttgtaca aatcgcgcgc 900 gccaccgacg aggccggccc gttggcccccctcgtgcgca ggtacatcga cgaggagctg 960 aagctgaagg ccgaggttcc gtcactacgcatcggcgacg atggccacct ctcgggtgga 1020 gagatcaacg gcgctgatgt ttttgccagcgtgcgcggca aggcatcctc accgcctgca 1080 gacattgagg tcggacccga ctcgaaccccacacttagct tcacgtcagg ctcggaaggc 1140 cggcctaagg gcgtgcttgg tcgacactacagcttggcca agtattttcg atggatggcc 1200 gagacgttcg gcatgggcga agagagccgcttcacactgc tctcgggtat cgcgcacgac 1260 cctgtgcagc gagatatctt cacgccactgtacctgggcg cgcgcctact ggtgccgtcc 1320 aaggagaata ttgcacacga gcgtctagcagagtggttca agcgctttga accaacagtg 1380 acacacctga cgccggccat gggtcagattttagtcggcg gcgctaccgc acagttccct 1440 gccctgaaaa cagcctactt cgtcggcgatgtgctgacga cgcgagactg ccgcagtctg 1500 cgtgagctcg cggcaaacgt tgacattgttaacatgtacg ggacgactga gacgagcaga 1560 gctgtcagct actacaagat cccgaaccgcgcctcagacc cggactttct ggagagattg 1620 ggcaaggaca caatccctgc tggaactggcatggaaaacg ttcagctttt ggttgtcaac 1680 cgggaagata ggacaaagct ttgtggtatcggcgaagtgg gcgagatcta cgtgcgtgcg 1740 gctggtctgg ctgagggcta caagggcgaccccgctttga acgaacagaa gttcctgatg 1800 aactggttcg tggataacaa caagtgggttgaggcggaca gggtgcaccc aaccaaggat 1860 gcggcatgga gaaagtacta caagggccctctcgacaggc tgtaccgcac aggtgacctc 1920 ggaaagtatt tggattcggg cgatgtggaatgcactggtc gtgcagacga tcaagtcaaa 1980 atcaggggct tcaggattga actcaacgacattgacagca acctgagtca gagctccctc 2040 atcagggact gcaagacgct tgtgcgaagggacaagaacg aggagccgaa gctggtcagc 2100 tacgttgtgc ctgagctcaa gcaatggccccaatggctca agcttcatgg ctacgaggat 2160 gctgaagacg atgagggcac gcaaattggagcaaccaagg tatacttcaa gaggtatcgt 2220 cgtatgcagg ctgagcttcg cgaccatctcaagtcaaggt tgccgaccta cgccgttcct 2280 agcatcttca ttgtcctgga gaagctgccattaaacccca acggcaaggt tgacaagccc 2340 aatctacctt ttcccgatat tgccgagcaaaccgcggaag cttcaagcga ggagattgag 2400 cgatgggagt ctttgaccga gactgagcgtgctgttgcca ccaggtgggc tgcgttgatc 2460 cagggtctga acgaaaagtc gatcgcgcccgataaccact tctttgacct cggcggtcac 2520 agtattctgg cacagcaaat gctgctcgacgtgcgcaagc agatgggtgc taatgtgtct 2580 atcaacaccc tttacgagaa ccccacgcttggggcattca gtcttcagat tgacaagcat 2640 cttggagcag ccaatgatgc tagcaccagccaagtcgagg atgaggcaaa ctcgtatgcc 2700 aaggctcgtg atgatctcgt taagaaacttccagcctcat acaagacagc agatccgtcg 2760 tcgatccggg cgtcatccag acctaccatcttcctgactg gcgcaacggg tttcttgggt 2820 gcctttttga tccgcgatat cctgcagaggacgagccgac agctgaagct catcgcacac 2880 gtgcgtgcca aggaccaaaa agcggcgacagagcgtctga cgcggtcact acagggctac 2940 ggtatctggc gcgacgagtg ggctgggcgcctttcctgcg tagtgggtga cctagccaag 3000 ccgcaacttg gtattgatca gcccacatgggagcgcctgt cgaacgaggt ggacgtagtt 3060 atccacaacg gtgcgacagt ccactgggtgcgccgctggc aagacatgct ggccgccaac 3120 gtcacatcga caatcgaggc catgcggctgtgcaacgagg gcaagccaaa gttgttcact 3180 ttcgtcagct caactagcgt cttggacactgagcactatg tgcagttgtc ggagaggcaa 3240 ctgagcaccg gccaaggcgc cgtccccgagtctgacgacc tcgaaggcag tgccactggc 3300 ttgggtacag gttacggcca aaccaacatcacgagtcagt ttaggaagcc tggctctggg 3360 agagctcccg cgccaaattg ccccagaaattaagcatcca tgccaagagc ggcttcccga 3420 tgtcgcaata ccgaatgtca ttgtggatcaatttttttta cattctgcag ggatctaatt 3480 gagctttgaa aagtcgagtc accgtcaccagagttactca ctcttctttt ttttcagcaa 3540 gcatcttggt gggctgctgg cttcttgttttgcttgatgt ctgtaaaaga ttcaatcgag 3600 gccaggggca gttttaagta catataaactttgcaaaaag ctagggttca cataccagtc 3660 aacgcataca cactggcgca gtagtaccccgccaattgtc ccccatcacg ataaatcgtc 3720 accatggcgc atcagctccc cgacccaacggtcgacctcg actggtctgg ctacgtcggc 3780 gccattcatg agatctttgc cactaatgcccagaagcacc cggagcgggt gtgcgtgatc 3840 gagacagagt cctccgaggc accggaaaggatatttacct acaagcagat ctttgaggcg 3900 tcaaatgtcc tggcgcacta cctacatgatgctggagtca ctaatggcga tgtggtcatg 3960 atctgggcgc ataggtcagt tgacctggttgtcagcatca tgggtgttct tgtaagctgc 4020 atcttccctc cctatgaact tgaccgattccccgccttgt cttagccagc actgtttctc 4080 cgagagaacg gaaacactga catgaccaaaaccattccta ggctgccgga gctacattca 4140 gtgtccttga cccattatac ccgccatctcgtcagcagat ctacctcgaa gtatccggcc 4200 cgaccgccct tgtacaaatc gcgcgcgccaccgacgaggc cggcccgttg gcccccctcg 4260 tgcgcaggta catcgacgag gagctgaagctgaaggccga ggttccgtca ctacgcatcg 4320 gcgacgatgg ccacctctcg ggtggagagatcaacggcgc tgatgttttt gccagcgtgc 4380 gcggcaaggc atcctcaccg cctgcagacattgaggtcgg acccgactcg aaccccacac 4440 ttagcttcac gtcaggctcg gaaggccggcctaagggcgt gcttggtcga cactacagct 4500 tggccaagta ttttcgatgg atggccgagacgttcggcat gggcgaagag agccgcttca 4560 cactgctctc gggtatcgcg cacgaccctgtgcagcgaga tatcttcacg ccactgtacc 4620 tgggcgcgcg cctactggtg ccgtccaaggagaatattgc acacgagcgt ctagcagagt 4680 ggttcaagcg ctttgaacca acagtgacacacctgacgcc ggccatgggt cagattttag 4740 tcggcggcgc taccgcacag ttccctgccctgaaaacagc ctacttcgtc ggcgatgtgc 4800 tgacgacgcg agactgccgc agtctgcgtgagctcgcggc aaacgttgac attgttaaca 4860 tgtacgggac gactgagacg agcagagctgtcagctacta caagatcccg aaccgcgcct 4920 cagacccgga ctttctggag agattgggcaaggacacaat ccctgctgga actggcatgg 4980 aaaacgttca gcttttggtt gtcaaccgggaagataggac aaagctttgt ggtatcggcg 5040 aagtgggcga gatctacgtg cgtgcggctggtctggctga gggctacaag ggcgaccccg 5100 ctttgaacga acagaagttc ctgatgaactggttcgtgga taacaacaag tgggttgagg 5160 cggacagggt gcacccaacc aaggatgcggcatggagaaa gtactacaag ggccctctcg 5220 acaggctgta ccgcacaggt gacctcggaaagtatttgga ttcgggcgat gtggaatgca 5280 ctggtcgtgc agacgatcaa gtcaaaatcaggggcttcag gattgaactc aacgacattg 5340 acagcaacct gagtcagagc tccctcatcagggactgcaa gacgcttgtg cgaagggaca 5400 agaacgagga gccgaagctg gtcagctacgttgtgcctga gctcaagcaa tggccccaat 5460 ggctcaagct tcatggctac gaggatgctgaagacgatga gggcacgcaa attggagcaa 5520 ccaaggtata cttcaagagg tatcgtcgtatgcaggctga gcttcgcgac catctcaagt 5580 caaggttgcc gacctacgcc gttcctagcatcttcattgt cctggagaag ctgccattaa 5640 accccaacgg caaggttgac aagcccaatctaccttttcc cgatattgcc gagcaaaccg 5700 cggaagcttc aagcgaggag attgagcgatgggagtcttt gaccgagact gagcgtgctg 5760 ttgccaccag gtgggctgcg ttgatccagggtctgaacga aaagtcgatc gcgcccgata 5820 accacttctt tgacctcggc ggtcacagtattctggcaca gcaaatgctg ctcgacgtgc 5880 gcaagcagat gggtgctaat gtgtctatcaacacccttta cgagaacccc acgcttgggg 5940 cattcagtct tcagattgac aagcatcttggagcagccaa tgatgctagc accagccaag 6000 tcgaggatga ggcaaactcg tatgccaaggctcgtgatga tctcgttaag aaacttccag 6060 cctcatacaa gacagcagat ccgtcgtcgatccgggcgtc atccagacct accatcttcc 6120 tgactggcgc aacgggtttc ttgggtgcctttttgatccg cgatatcctg cagaggacga 6180 gccgacagct gaagctcatc gcacacgtgcgtgccaagga ccaaaaagcg gcgacagagc 6240 gtctgacgcg gtcactacag ggctacggtatctggcgcga cgagtgggct gggcgccttt 6300 cctgcgtagt gggtgaccta gccaagccgcaacttggtat tgatcagccc acatgggagc 6360 gcctgtcgaa cgaggtggac gtagttatccacaacggtgc gacagtccac tgggtgcgcc 6420 gctggcaaga catgctggcc gccaacgtcacatcgacaat cgaggccatg cggctgtgca 6480 acgagggcaa gccaaagttg ttcactttcgtcagctcaac tagcgtcttg gacactgagc 6540 actatgtgca gttgtcggag aggcaactgagcaccggcca aggcgccgtc cccgagtctg 6600 acgacctcga aggcagtgcc actggcttgggtacaggtta cggccaaacc aagtggatct 6660 cggagcagct cgtcagggag gcgggccgacgcggacttcg aggctccgtt gtcaggccag 6720 gctacattct gggagatttc gagtcgggatgttccaacac agacgacttc ctcattcgct 6780 tcctcaaggg ctgtatccag ctcggcacgaggccccgcat tctcaatact gtcaatgccg 6840 tgcccgtcaa ccacgttgcg cgtgttgttgtcgcggctgg tctcaaccct gtacccgtcc 6900 agggtaatga aggtgtccac gtggtccacgttacgggcca cccgcgcctg cggatgaacg 6960 agtatctctc gttgcttgag ttctacggctacaaggtgcc cgaggtgccg tatgattcat 7020 ggaaggagga gctggagcag tacgtgtctgcgggcgcggg tgtcgagcgc gaccaggagc 7080 agcacgcgct gctacctctc ttccacctctgtatctcgga cctgcccgcc aacactaagg 7140 cacctgagct tgaggaccaa aacgctgtcgcggtcctcaa ggcggacgct gaggcctgga 7200 ccgggctcga cgagagcgcg ggctacggcatcggcaggga ggacgtcgga aggtacctcc 7260 gctacctagc catgatcaag tttgtcccctggcctacgtc gaggggcagg cctttgcctg 7320 aggtcagcat cagcacggag caggtggctgctatgggtgc aggcgtcggt ggacgtggtg 7380 gtgcgggtgc gggacagtga acgaaaagaatggtggacgg ctagccatct gagtatatga 7440 ctgttttctt taaatgaatt ggtagctttggctttttaaa aggtgcttgg tttggaattt 7500 aaagttgatt tctcgggcta aaaaaaaaaaaaaaaactcg agggggggcc cggtacccaa 7560 ttcgccctat agtgagtcgt attcaa 75863 1188 PRT Magnaporthe grisea 3 Met Ala His Gln Leu Pro Asp Pro Thr ValAsp Leu Asp Trp Ser Gly 1 5 10 15 Tyr Val Gly Ala Ile His Glu Ile PheAla Thr Asn Ala Gln Lys His 20 25 30 Pro Glu Arg Val Cys Val Ile Glu ThrGlu Ser Ser Glu Ala Pro Glu 35 40 45 Arg Ile Phe Thr Tyr Lys Gln Ile PheGlu Ala Ser Asn Val Leu Ala 50 55 60 His Tyr Leu His Asp Ala Gly Val ThrAsn Gly Asp Val Val Met Ile 65 70 75 80 Trp Ala His Arg Ser Val Asp LeuVal Val Ser Ile Met Gly Val Leu 85 90 95 Ala Ala Gly Ala Thr Phe Ser ValLeu Asp Pro Leu Tyr Pro Pro Ser 100 105 110 Arg Gln Gln Ile Tyr Leu GluVal Ser Gly Pro Thr Ala Leu Val Gln 115 120 125 Ile Ala Arg Ala Thr AspGlu Ala Gly Pro Leu Ala Pro Leu Val Arg 130 135 140 Arg Tyr Ile Asp GluGlu Leu Lys Leu Lys Ala Glu Val Pro Ser Leu 145 150 155 160 Arg Ile GlyAsp Asp Gly His Leu Ser Gly Gly Glu Ile Asn Gly Ala 165 170 175 Asp ValPhe Ala Ser Val Arg Gly Lys Ala Ser Ser Pro Pro Ala Asp 180 185 190 IleGlu Val Gly Pro Asp Ser Asn Pro Thr Leu Ser Phe Thr Ser Gly 195 200 205Ser Glu Gly Arg Pro Lys Gly Val Leu Gly Arg His Tyr Ser Leu Ala 210 215220 Lys Tyr Phe Arg Trp Met Ala Glu Thr Phe Gly Met Gly Glu Glu Ser 225230 235 240 Arg Phe Thr Leu Leu Ser Gly Ile Ala His Asp Pro Val Gln ArgAsp 245 250 255 Ile Phe Thr Pro Leu Tyr Leu Gly Ala Arg Leu Leu Val ProSer Lys 260 265 270 Glu Asn Ile Ala His Glu Arg Leu Ala Glu Trp Phe LysArg Phe Glu 275 280 285 Pro Thr Val Thr His Leu Thr Pro Ala Met Gly GlnIle Leu Val Gly 290 295 300 Gly Ala Thr Ala Gln Phe Pro Ala Leu Lys ThrAla Tyr Phe Val Gly 305 310 315 320 Asp Val Leu Thr Thr Arg Asp Cys ArgSer Leu Arg Glu Leu Ala Ala 325 330 335 Asn Val Asp Ile Val Asn Met TyrGly Thr Thr Glu Thr Ser Arg Ala 340 345 350 Val Ser Tyr Tyr Lys Ile ProAsn Arg Ala Ser Asp Pro Asp Phe Leu 355 360 365 Glu Arg Leu Gly Lys AspThr Ile Pro Ala Gly Thr Gly Met Glu Asn 370 375 380 Val Gln Leu Leu ValVal Asn Arg Glu Asp Arg Thr Lys Leu Cys Gly 385 390 395 400 Ile Gly GluVal Gly Glu Ile Tyr Val Arg Ala Ala Gly Leu Ala Glu 405 410 415 Gly TyrLys Gly Asp Pro Ala Leu Asn Glu Gln Lys Phe Leu Met Asn 420 425 430 TrpPhe Val Asp Asn Asn Lys Trp Val Glu Ala Asp Arg Val His Pro 435 440 445Thr Lys Asp Ala Ala Trp Arg Lys Tyr Tyr Lys Gly Pro Leu Asp Arg 450 455460 Leu Tyr Arg Thr Gly Asp Leu Gly Lys Tyr Leu Asp Ser Gly Asp Val 465470 475 480 Glu Cys Thr Gly Arg Ala Asp Asp Gln Val Lys Ile Arg Gly PheArg 485 490 495 Ile Glu Leu Asn Asp Ile Asp Ser Asn Leu Ser Gln Ser SerLeu Ile 500 505 510 Arg Asp Cys Lys Thr Leu Val Arg Arg Asp Lys Asn GluGlu Pro Lys 515 520 525 Leu Val Ser Tyr Val Val Pro Glu Leu Lys Gln TrpPro Gln Trp Leu 530 535 540 Lys Leu His Gly Tyr Glu Asp Ala Glu Asp AspGlu Gly Thr Gln Ile 545 550 555 560 Gly Ala Thr Lys Val Tyr Phe Lys ArgTyr Arg Arg Met Gln Ala Glu 565 570 575 Leu Arg Asp His Leu Lys Ser ArgLeu Pro Thr Tyr Ala Val Pro Ser 580 585 590 Ile Phe Ile Val Leu Glu LysLeu Pro Leu Asn Pro Asn Gly Lys Val 595 600 605 Asp Lys Pro Asn Leu ProPhe Pro Asp Ile Ala Glu Gln Thr Ala Glu 610 615 620 Ala Ser Ser Glu GluIle Glu Arg Trp Glu Ser Leu Thr Glu Thr Glu 625 630 635 640 Arg Ala ValAla Thr Arg Trp Ala Ala Leu Ile Gln Gly Leu Asn Glu 645 650 655 Lys SerIle Ala Pro Asp Asn His Phe Phe Asp Leu Gly Gly His Ser 660 665 670 IleLeu Ala Gln Gln Met Leu Leu Asp Val Arg Lys Gln Met Gly Ala 675 680 685Asn Val Ser Ile Asn Thr Leu Tyr Glu Asn Pro Thr Leu Gly Ala Phe 690 695700 Ser Leu Gln Ile Asp Lys His Leu Gly Ala Ala Asn Asp Ala Ser Thr 705710 715 720 Ser Gln Val Glu Asp Glu Ala Asn Ser Tyr Ala Lys Ala Arg AspAsp 725 730 735 Leu Val Lys Lys Leu Pro Ala Ser Tyr Lys Thr Ala Asp ProSer Ser 740 745 750 Ile Arg Ala Ser Ser Arg Pro Thr Ile Phe Leu Thr GlyAla Thr Gly 755 760 765 Phe Leu Gly Ala Phe Leu Ile Arg Asp Ile Leu GlnArg Thr Ser Arg 770 775 780 Gln Leu Lys Leu Ile Ala His Val Arg Ala LysAsp Gln Lys Ala Ala 785 790 795 800 Thr Glu Arg Leu Thr Arg Ser Leu GlnGly Tyr Gly Ile Trp Arg Asp 805 810 815 Glu Trp Ala Gly Arg Leu Ser CysVal Val Gly Asp Leu Ala Lys Pro 820 825 830 Gln Leu Gly Ile Asp Gln ProThr Trp Glu Arg Leu Ser Asn Glu Val 835 840 845 Asp Val Val Ile His AsnGly Ala Thr Val His Trp Val Arg Arg Trp 850 855 860 Gln Asp Met Leu AlaAla Asn Val Thr Ser Thr Ile Glu Ala Met Arg 865 870 875 880 Leu Cys AsnGlu Gly Lys Pro Lys Leu Phe Thr Phe Val Ser Ser Thr 885 890 895 Ser ValLeu Asp Thr Glu His Tyr Val Gln Leu Ser Glu Arg Gln Leu 900 905 910 SerThr Gly Gln Gly Ala Val Pro Glu Ser Asp Asp Leu Glu Gly Ser 915 920 925Ala Thr Gly Leu Gly Thr Gly Tyr Gly Gln Thr Lys Trp Ile Ser Glu 930 935940 Gln Leu Val Arg Glu Ala Gly Arg Arg Gly Leu Arg Gly Ser Val Val 945950 955 960 Arg Pro Gly Tyr Ile Leu Gly Asp Phe Glu Ser Gly Cys Ser AsnThr 965 970 975 Asp Asp Phe Leu Ile Arg Phe Leu Lys Gly Cys Ile Gln LeuGly Thr 980 985 990 Arg Pro Arg Ile Leu Asn Thr Val Asn Ala Val Pro ValAsn His Val 995 1000 1005 Ala Arg Val Val Val Ala Ala Gly Leu Asn ProVal Pro Val Gln 1010 1015 1020 Gly Asn Glu Gly Val His Val Val His ValThr Gly His Pro Arg 1025 1030 1035 Leu Arg Met Asn Glu Tyr Leu Ser LeuLeu Glu Phe Tyr Gly Tyr 1040 1045 1050 Lys Val Pro Glu Val Pro Tyr AspSer Trp Lys Glu Glu Leu Glu 1055 1060 1065 Gln Tyr Val Ser Ala Gly AlaGly Val Glu Arg Asp Gln Glu Gln 1070 1075 1080 His Ala Leu Leu Pro LeuPhe His Leu Cys Ile Ser Asp Leu Pro 1085 1090 1095 Ala Asn Thr Lys AlaPro Glu Leu Glu Asp Gln Asn Ala Val Ala 1100 1105 1110 Val Leu Lys AlaAsp Ala Glu Ala Trp Thr Gly Leu Asp Glu Ser 1115 1120 1125 Ala Gly TyrGly Ile Gly Arg Glu Asp Val Gly Arg Tyr Leu Arg 1130 1135 1140 Tyr LeuAla Met Ile Lys Phe Val Pro Trp Pro Thr Ser Arg Gly 1145 1150 1155 ArgPro Leu Pro Glu Val Ser Ile Ser Thr Glu Gln Val Ala Ala 1160 1165 1170Met Gly Ala Gly Val Gly Gly Arg Gly Gly Ala Gly Ala Gly Gln 1175 11801185

What is claimed is:
 1. A method for identifying a test compound as acandidate for an antibiotic, comprising: a) contacting an α-AminoadipateReductase polypeptide with said test compound; and b) detecting thepresence or absence of binding between said test compound and saidα-Aminoadipate Reductase polypeptide; wherein binding indicates thatsaid test compound is a candidate for an antibiotic.
 2. The method ofclaim 1, wherein said α-Aminoadipate Reductase polypeptide is a fungalα-Aminoadipate Reductase polypeptide.
 3. The method of claim 1, whereinsaid α-Aminoadipate Reductase polypeptide is a Magnaportheα-Aminoadipate Reductase polypeptide .
 4. The method of claim 1, whereinsaid α-Aminoadipate Reductase polypeptide is SEQ ID NO:
 3. 5. A methodfor determining whether a compound identified as an antibiotic candidateby the method of claim 1 has antifungal activity, further comprising:contacting a fungus or fungal cells with said antibiotic candidate anddetecting the decrease in growth, viability, or pathogenicity of saidfungus or fungal cells.
 6. A method for identifying a test compound as acandidate for an antibiotic, comprising: a) contacting said testcompound with at least one polypeptide selected from the groupconsisting of: a polypeptide having at least ten consecutive amino acidsof a fungal α-Aminoadipate Reductase, a polypeptide having at least 50%sequence identity with a fungal α-Aminoadipate Reductase, and apolypeptide having at least 10% of the activity thereof; and b)detecting the presence and/or absence of binding between said testcompound and said polypeptide; wherein binding indicates that said testcompound is a candidate for an antibiotic.
 7. A method for determiningwhether a compound identified as an antibiotic candidate by the methodof claim 6 has antifungal activity, further comprising: contacting afungus or fungal cells with said antibiotic candidate and detecting adecrease in growth, viability, or pathogenicity of said fungus or fungalcells.
 8. A method for identifying a test compound as a candidate for anantibiotic, comprising: a) contacting L-2-Aminoadipate and NADPH and ATPwith an α-Aminoadipate Reductase; b) contacting L-2-Aminoadipate andNADPH and ATP with α-Aminoadipate Reductase and said test compound; andc) determining the change in concentration for at least one of thefollowing: L-2-Aminoadipate, L-2-Aminoadipate 6-semialdehyde, NADP+,NADPH, AMP, ATP, H₂O and/or pyrophosphate; wherein a change inconcentration for any of the above substances between steps (a) and (b)indicates that said test compound is a candidate for an antibiotic. 9.The method of claim 8, wherein said α-Aminoadipate Reductase is a fungalα-Aminoadipate Reductase.
 10. The method of claim 8, wherein saidα-Aminoadipate Reductase is a Magnaporthe α-Aminoadipate Reductase. 11.The method of claim 8, wherein said α-Aminoadipate Reductase is SEQ IDNO:
 3. 12. A method for determining whether a compound identified as anantibiotic candidate by the method of claim 8 has antifungal activity,further comprising: contacting a fungus or fungal cells with saidantibiotic candidate and detecting a decrease in growth, viability, orpathogenicity of said fungus or fungal cells.
 13. A method foridentifying a test compound as a candidate for an antibiotic,comprising: a) contacting L-2-Aminoadipate 6-semialdehyde, NADP+, AMP,pyrophosphate, and H₂O with an α-Aminoadipate Reductase; b) contactingL-2-Aminoadipate 6-semialdehyde, NADP+, AMP, pyrophosphate, and, H₂Owith an α-Aminoadipate Reductase and said test compound; and c)determining the change in concentration for at least one of thefollowing: L-2-Aminoadipate, L-2-Aminoadipate 6-semialdehyde, NADP+,NADPH, AMP, ATP, H₂O and/or pyrophosphate; wherein a change inconcentration for any of the above substances between steps (a) and (b)indicates that said test compound is a candidate for an antibiotic. 14.The method of claim 13, wherein said α-Aminoadipate Reductase is afungal α-Aminoadipate Reductase.
 15. The method of claim 13, whereinsaid α-Aminoadipate Reductase is a Magnaporthe α-Aminoadipate Reductase.16. The method of claim 13, wherein said α-Aminoadipate Reductase is SEQID NO:
 3. 17. A method for determining whether a compound identified asan antibiotic candidate by the method of claim 13 has antifungalactivity, further comprising: contacting a fungus or fungal cells withsaid antibiotic candidate and detecting a decrease in growth, viability,or pathogenicity of said fungus or flingal cells.
 18. A method foridentifying a test compound as a candidate for an antibiotic,comprising: a) contacting L-2-Aminoadipate and NADPH and ATP with apolypeptide selected from the group consisting of: a polypeptide havingat least 50% sequence identity with an α-Aminoadipate Reductase, apolypeptide having at least 50% sequence identity with an α-AminoadipateReductase and having at least 10% of the activity thereof, and apolypeptide comprising at least 100 consecutive amino acids of anα-Aminoadipate Reductase b) contacting L-2-Aminoadipate and NADPH andATP with said polypeptide and said test compound; and c) determining thechange in concentration for at least one of the following:L-2-Aminoadipate, L-2-Aminoadipate 6-semialdehyde, NADP+, NADPH, AMP,ATP, H₂O and/or pyrophosphate; wherein a change in concentration for anyof the above substances between steps (a) and (b) indicates that saidtest compound is a candidate for an antibiotic.
 19. A method foridentifying a test compound as a candidate for an antibiotic,comprising: a) contacting L-2-Aminoadipate 6-semialdehyde, NADP+, AMP,pyrophosphate, and H₂O with a polypeptide selected from the groupconsisting of: a polypeptide having at least 50% sequence identity withan α-Aminoadipate Reductase, a polypeptide having at least 50% sequenceidentity with an α-Aminoadipate Reductase and at least 10% of theactivity thereof, and a polypeptide comprising at least 100 consecutiveamino acids of an α-Aminoadipate Reductase b) contactingL-2-Aminoadipate 6-semialdehyde, NADP+, AMP, pyrophosphate, and H₂O,with said polypeptide and said test compound; and c) determining thechange in concentration for at least one of the following:L-2-Aminoadipate, L-2-Aminoadipate 6-semialdehyde, NADP+, NADPH, AMP,ATP, H₂O and/or pyrophosphate; wherein a change in concentration for anyof the above substances between steps (a) and (b) indicates that saidtest compound is a candidate for an antibiotic.
 20. A method foridentifying a test compound as a candidate for an antibiotic,comprising: a) measuring the expression of an α-Aminoadipate Reductasein a cell, cells, tissue, or an organism in the absence of saidcompound; b) contacting said cell, cells, tissue, or organism with saidtest compound and measuring the expression of said α-AminoadipateReductase in said fungus or fungal cell; c) comparing the expression ofα-Aminoadipate Reductase in steps (a) and (b); wherein a lowerexpression in the presence of said test compound indicates that saidcompound is a candidate for an antibiotic.
 21. The method of claim 20wherein said a cell, cells, tissue, or organism is, or is derived from afungus.
 22. The method of claim 20 wherein said cell, cells, tissue, ororganism is, or is derived from a Magnaporthe fungus or fungal cell. 23.The method of claim 20, wherein said α-Aminoadipate Reductase is SEQ IDNO:
 3. 24. The method of claim 20, wherein the expression ofα-Aminoadipate Reductase is measured by detecting AAR1 mRNA.
 25. Themethod of claim 20, wherein the expression of α-Aminoadipate Reductaseis measured by detecting α-Aminoadipate Reductase polypeptide.
 26. Amethod for identifying a test compound as a candidate for an antibiotic,comprising: a) providing cells having one form of an α-AminoadipateReductase gene, and providing comparison cells having a different formof an α-Aminoadipate Reductase gene, b) contacting said cells and saidcomparison cells with a test compound and determining the growth of saidcells and comparison cells in the presence of the test compound; whereina difference in growth between said cells and said comparison cells inthe presence of said compound indicates that said compound is acandidate for an antibiotic.
 27. The method of claim 26 wherein thecells are fungal cells.
 28. The method of claim 26 wherein the cells areMagnaporthe cells.
 29. The method of claim 26 wherein said form and saidcomparison form of the α-Aminoadipate Reductase are fungalα-Aminoadipate Reductases.
 30. The method of claim 26, wherein at leastone form is a Magnaporthe α-Aminoadipate Reductase.
 31. The method ofclaim 26 wherein said form and said comparison form of theα-Aminoadipate Reductase are non-fungal α-Aminoadipate Reductases. 32.The method of claim 26 wherein one form of the α-Aminoadipate Reductaseis a fungal α-Aminoadipate Reductase, and the other form is a non-fungalα-Aminoadipate Reductase.
 33. A method for identifying a test compoundas a candidate for an antibiotic, comprising: d) providing cells havingone form of a gene in the lysine biochemical and/or genetic pathway andproviding comparison cells having a different form of said gene. e)contacting said cells and comparison cells with a said test compound, f)determining the growth of said cells and comparison cells in thepresence of said test compound; wherein a difference in growth betweensaid cells and said comparison cells in the presence of said compoundindicates that said compound is a candidate for an antibiotic.
 34. Themethod of claim 33 wherein the cells are fungal cells.
 35. The method ofclaim 33 wherein the cells are Magnaporthe cells.
 36. The method ofclaim 33 wherein said form and said comparison form of the lysinebiosynthesis gene are fungal lysine biosynthesis genes.
 37. The methodof claim 33, wherein at least one form is a Magnaporthe lysinebiosynthesis gene.
 38. The method of claim 33 wherein said form and saidcomparison form of the lysine biosynthesis genes are non-fungal lysinebiosynthesis genes.
 39. The method of claim 33 wherein one form of thelysine biosynthesis gene is a fungal lysine biosynthesis gene, and theother form is a non-fungal lysine biosynthesis gene.
 40. A method fordetermining whether a test compound identified as an antibioticcandidate by the method of claim 33 has antifungal activity, furthercomprising: contacting a fungus or fungal cells with said antibioticcandidate and detecting a decrease in growth, viability, orpathogenicity of said fungus or fungal cells.
 41. A method foridentifying a test compound as a candidate for an antibiotic,comprising: (a) providing paired growth media; comprising a first mediumand a second medium, wherein said second medium contains a higher levelof lysine than said first medium; (b) contacting an organism with saidtest compound; (c) inoculating said first and second media with saidorganism; and (d) determining the growth of said organism; wherein adifference in growth of the organism between said first and second mediaindicates that said test compound is a candidate for an antibiotic. 42.The method of claim 41, wherein said organism is a fungus.
 43. Themethod of claim 41, wherein said organism is Magnaporthe.
 44. Anisolated polynucleotide comprising a nucleotide sequence that encodes apolypeptide of SEQ ID NO:
 3. 45. The polynucleotide of claim 44comprising the nucleotide sequence of SEQ ID NO:
 1. 46. An expressioncassette comprising the polynucleotide of claim
 45. 47. The isolatedpolynucleotide of claim 44 comprising a nucleotide sequence of at least50 to at least 95% sequence identity to SEQ ID NO:
 1. 48. A polypeptideconsisting essentially of the amino acid sequence of SEQ ID NO:
 3. 49. Apolypeptide comprising the amino acid sequence of SEQ ID NO: 3.